Compositions for the diagnosis and treatment of Chediak-Higashi syndrome

ABSTRACT

The present invention relates to the identification of novel nucleic acid molecules and proteins encoded by such nucleic acid molecules or degenerate variants thereof, that participate in the differentiation and/or function of intracellular vesicles. The nucleic acid molecules of the present invention represent the genes corresponding to the mammalian bg gene, a gene that, when mutated, is responsible for the human Chediak-Higashi syndrome.

This application claims priority under 35 U.S.C. § 119(e) to U.S. provisonal application Serial No. 60/021,064, filed Jul. 1, 1996, U.S. provisonal application Serial No. 60/015,673, filed Apr. 19, 1996 and U.S. provisonal application Serial No. 60/013,883, filed Mar. 22, 1996, each of which is incorporated herein by reference in its entirety.

1. INTRODUCTION

The present invention relates to the identification of novel nucleic acid molecules and proteins encoded by such nucleic acid molecules or degenerate, especially naturally occurring, variants thereof, that, when mutated, lead to disorders involving abnormal intracellular vesicles, especially abnormal lysosomes, melanosomes, platelet dense granules and cytolytic granules, including Chediak-Higashi syndrome (CHS). The nucleic acid molecules of the present invention represent the genes corresponding to the mammalian bg gene, including the human bg gene, which are involved in the normal differentiation and/or function of such intracellular vesicles. Nucleic acid molecules representing loss-of-function alleles of the human bg gene bring about Chediak-Higashi syndrome (CHS), in individuals homozygous for such alleles.

In particular, the compositions of the present invention include nucleic acid molecules (e.g., bg gene), including recombinant DNA molecules, cloned genes or degenerate, especially naturally occurring, variants thereof, which encode novel bg gene products, and antibodies directed against such bg gene products or conserved variants or fragments thereof. The compositions of the present invention additionally include cloning vectors, including expression vectors, containing the nucleic acid molecules of the invention and hosts which have been transformed with such nucleic acid molecules.

In addition, this invention presents methods for the diagnostic evaluation and prognosis of disorders involving abnormal intracellular vesicles, especially abnormal lysosomes, melanosomes, platelet dense granules and cytolytic granules, including CHS, and for the identification of subjects having a predisposition to such conditions. For example, nucleic acid molecules of the invention can be used as diagnostic hybridization probes or as primers for diagnostic PCR analysis for the identification of bg gene mutations, allelic variations and regulatory defects in the bg gene.

Further, methods and compositions are presented for the treatment of disorders involving abnormal intracellular vesicles, especially abnormal lysosomes, melanosomes, platelet dense granules and cytolytic granules, including CHS. Such methods and compositions are capable of modulating the level of bg gene expression and/or the level of bg gene product activity.

Still further, the present invention relates to methods for the use of the bg gene, bg gene products and/or cells expressing wild type or mutant bg gene sequences for the identification of compounds which modulate bg gene expression and/or the activity of bg gene products. Such compounds can be used as agents to control disorders involving abnormal intracellular vesicles, especially abnormal lysosomes, melanosomes, platelet dense granules and cytolytic granules, in particular, therapeutic agents in the treatment of CHS.

2. BACKGROUND OF THE INVENTION

Chediak-Higashi syndrome (CHS) is a lethal autosomal recessive disorder of humans mapping to 1q43. The clinical manifestations of this disorder include hypopigmentation, defective immune cell function, including severely impaired natural killer cell activity, and defective antibody-dependent, lymphocyte-mediated cytolysis against tumor cell targets. Further, neural degeneration is observed and, finally, the occurrence of a mononuclear cell lymphoma develops, which causes the death of afflicted individuals.

As mentioned above, the disease is accompanied by a marked susceptibility to infections. Young children have repeated infections, usually with gram-positive organisms of the staphylococcal and streptococcal type. Further, during the course of the disease, children may develop a progressive peripheral neuropathy. Children surviving the early infectious episodes (8-18 years of age), most frequently develop terminal lymphoreticular malignancy. Few patients survive beyond twenty years.

Pathological manifestation of the syndrome includes enlarged vesicles affecting lysosomes, melanosomes, platelet dense granules, cytolytic granules and Schwann cell granules. The abnormal size of these vesicles is thought to result from a malregulation of vesicle fusion or fission. Abnormal membrane-bound lysosomal-like organelles have been found in cells of the buccal mucosa, Schwann cells, pancreas, liver, gastric and duodenal mucosa, adrenal, pituitary, spleen, kidney, bone marrow, hair skin, iris and conjunctiva. The giant granules observed resemble the normal granules of the specific cell type in both fine structure and cytochemic reactions and result from the fusion of small primary granules.

Similar phenotypes are found in other species, most notably the beige mouse and the Aleutian mink, but are also found in such species as the Persian cat, cattle and even the killer whale. Somatic cell fusion studies have suggested that mutations within the same gene in mouse, mink, and man were responsible for the CHS-like phenotype in each of these species. In mice, the gene responsible for such a phenotype is the beige (bg) gene. Such studies, however, were not able to elucidate either the function or the identity of the bg gene product.

Over the past thirty years numerous theories have been evoked to explain the nature of these disorders. For example, it has been suggested that the defect might be caused by alterations in membrane fluidity, defects in microtubules or microtubule associated proteins, or changes in cyclic nucleotides levels. Upon further examination, though, each of these theories has been found to be inadequate, thus highlighting the fact that a great need remains for the discovery of the causative agent of the lethal Chediak-Higashi syndrome genetic disorder.

3. SUMMARY OF THE INVENTION

The present invention relates to the identification of novel nucleic acid molecules and proteins encoded by such nucleic acid molecules or degenerate, especially naturally occurring, variants thereof, that, when mutated, lead to disorders involving abnormal intracellular vesicles, especially abnormal lysosomes, melanosomes, platelet dense granules and cytolytic granules, including Chediak-Higashi syndrome (CHS). The nucleic acid molecules of the present invention represent the genes corresponding to the mammalian bg gene, including the human bg gene, which are involved in the normal differentiation and/or function of such intracellular vesicles. Nucleic acid molecules representing loss-of-function alleles of the human bg gene bring about Chediak-Higashi syndrome (CHS), in individuals homozygous for such alleles.

In particular, the compositions of the present invention include nucleic acid molecules (e.g., bg gene), including recombinant DNA molecules, cloned genes or degenerate, especially naturally occurring, variants thereof, which encode novel bg gene products, and antibodies directed against such bg gene products or conserved variants or fragments thereof. The compositions of the present invention additionally include cloning vectors, including expression vectors, containing the nucleic acid molecules of the invention and hosts which have been transformed with such nucleic acid molecules.

Nucleic acid sequences of wild type and mutant forms of the murine bg gene are provided. Wild type murine bg gene produces a transcript of approximately 12-14 kb. The amino acid sequence of the predicted bg gene product indicates that the protein is novel.

Nucleic acid sequences of wild type forms of the human bg gene are also provided. The human bg gene produces alternatively spliced transcripts. The long, putatively full length bg transcript encodes a bg protein of 3801 amino acid residues, as shown in FIG. 7. A short form, alternatively spliced, human bg transcript encodes a bg protein of 3672 amino acid residues, as shown in FIG. 8. The amino acid sequence of the predicted human bg gene products indicates that the proteins are novel.

In addition, this invention presents methods for the diagnostic evaluation and prognosis of disorders involving abnormal intracellular vesicles, especially abnormal lysosomes, melanosomes, platelet dense granules and cytolytic granules, including CHS, and for the identification of subjects having a predisposition to such conditions. For example, nucleic acid molecules of the invention can be used as diagnostic hybridization probes or as primers for diagnostic PCR analysis for the identification of bg gene mutations, allelic variations and regulatory defects in the bg gene.

Further, methods and compositions are presented for the treatment of disorders involving abnormal intracellular vesicles, especially abnormal lysosomes, melanosomes, platelet dense granules and cytolytic granules, including CHS. Such methods and compositions are capable of modulating the level of bg gene expression and/or the level of bg gene product activity.

Still further, the present invention relates to methods for the use of the bg gene, bg gene products and/or cells expressing wild type or mutant bg gene sequences for the identification of compounds which modulate bg gene expression and/or the activity of bg gene products. Such compounds can be used as agents to control disorders involving abnormal intracellular vesicles, especially abnormal lysosomes, melanosomes, platelet dense granules and cytolytic granules, in particular, therapeutic agents in the treatment of CHS.

This invention is based, in part, on a combination of in vitro complementation using yeast artificial chromosomes (YACs), positional cloning techniques and mutation detection which, together, were used to successfully identify and clone the murine bg gene, as described in the Examples, below, presented in Sections 6-9. Such analyses included the identification and sequencing of two independent bg mutations, one an insertion of 117 base pairs and the other a point mutation which results in an in-frame, premature stop codon. Both mutations result in the product of transcripts encoding truncated BG proteins.

4. DESCRIPTION OF THE FIGURES

FIG. 1. Genetic and physical map of mouse chromosome 13 region containing the bg gene interval.

FIGS. 2A-2B. Diagram depicting yeast artificial chromosomes (YACs) spanning the minimal bg interval. Ability of YACs to complement the bg mutation is noted.

FIG. 3A. Wild type and bg mouse fibroblasts plated together to demonstrate differences in phenotypes between the two cell types. The arrows denote two wild type cells. Two bg cells are just below the indicated wild type cells. Note the difference in lysosome size and distribution. Magnification was approximately 500×.

FIG. 3B. The initial mixed isolate of complemented colony 195-4, isolated from 400 μg/ml G418. This colony, as isolated from the plate contained complemented and uncomplemented bg cells. Magnification was approximately 500×.

FIG. 3C. Colony 195-4 after 10 days in 800 μg/ml G418. Note that the colony after this period of time was homogeneously complemented (i.e., all of the bg cells appeared wild type with respect to the lysosomal morphology.) Magnification was approximately 500×.

FIG. 3D. Colony 195-4 after culture i 800 μg/ml G418 for ten days, then cultured without G418 for thirty days. The result illustrated here demonstrates that the YAC was responsible for the complementation, in that, when the cells were cultured without G418, they lost the YAC and reverted back to the mutant bg morphology. Magnification was approximately 500×.

FIG. 4. Nucleotide sequence (bottom line; SEQ ID NO:1) and amino acid sequence (top line; SEQ ID NO:2) of the 22B/30B gene (the murine bg gene).

FIGS. 5A-5D. Southern blot analysis of a chromosomal rearrangement associated with the bg allele. Southern blot analysis of a 510 bp fragment of 22B/30B hybridized to lane (1) C57BL/6J; (2) C57BL/6J-bg; (3) C57BL/6J-bg^(J) ; (4) C57BL/6J-bg^(10J) ; (5) C57BL/6J-bg^(11J) ; (6) C3H/HeJ; (7) C3H/H3J-bg^(2J) ; (8) DBA/2J; and (9)DBA/2J-CO-bg^(8J) DNAs digested with 5A: HindIII; 5B: PstI; 5C: BglII; and 5D: TaqI. Size markers are indicated.

FIG. 6. Diagram illustrating the location and structure of a Line 1 insertion representing mutation within the bg gene yielding truncated BG proteins which leads to a mutant bg phenotype (SEQ ID NO:8).

FIG. 7. Human long form (putative full length) bg gene nucleotide (bottom line) (SEQ ID NO:9) and derived amino acid (top line) (SEQ ID NO:10) sequences.

FIG. 8. Human short form, alternatively spliced bg gene nucleotide (bottom line) (SEQ ID NO:11)and derived amino acid (top line) (SEQ ID NO:12) sequences.

5. DETAILED DESCRIPTION OF THE INVENTION

Described herein are novel mammalian genes, the beige (bg) genes, including the human bg gene. Such genes are involved in the normal differentiation and/or function of intracellular vesicles. When such sequences are mutated such that, for example, a functional beige gene product (BG) is no longer produced, disorders develop involving abnormal intracellular vesicles, especially abnormal lysosomes, melanosomes, platelet dense granules and cytolytic granules, including Chediak-Higashi syndrome. Also described are recombinant mammalian, including human, bg DNA molecules, cloned genes, or degenerate variants thereof. The compositions of the present invention further include bg gene products (e.g., proteins) that are encoded by the hg gene, and the modulation of bg gene expression and/or bg gene product activity in the treatment of disorders involving abnormal intracellular vesicles, including, but not limited to CHS. Also described herein are antibodies against bg gene products (e.g., proteins), or conserved variants or fragments thereof, and nucleic acid probes useful for the identification of bg gene mutations and the use of such nucleic acid probes in, for example, the identification of individuals predisposed to such disorders and/or individuals who carry mutant bg alleles. Further described are methods for the use of the bg gene and/or bg gene products in the identification of compounds which modulate the activity of the hg gene product.

Murine bg nucleic acid and amino acid compositions of the invention are demonstrated in the Examples presented, below, in Sections 6 through 9. A gene, referred to herein as the 22B/30B gene, representing a candidate for the murine bg gene was identified via a combination of genetic and physical mapping coupled with a yeast artificial chromosome (YAC) complementation assay by which complementation of the bg mutation was assessed via analysis of the morphological phenotype of YAC-transformed bg fibroblasts. Identification and sequencing of two independent bg mutations revealed that the mutations resided within the 22B/30B gene, representing compelling evidence that the 22B/30B gene was the bg gene. For clarity, it should, therefore, be noted that the murine bg gene is also referred to herein as the 22B/30B gene.

Human bg nucleic acid and amino acid compositions of the invention are demonstrated in Example 10, below.

5.1. THE bg GENE

The bg gene, murine nucleic acid sequence of which is shown in FIG. 4 (SEQ ID NO:1) and human nucleic acid sequences of which are shown in FIGS. 7 and 8, is a novel gene involved in the normal differentiation and/or function of intracellular vesicles. Nucleic acid sequences of the bg gene are described herein. As used herein, "bg gene" refers to (a) a gene containing the DNA sequence shown in FIG. 4, FIG. 7 or FIG. 8; (b) any DNA sequence that encodes the amino acid sequence shown in FIG. 4 (SEQ ID NO:2), FIG. 7 or FIG. 8; (c) any DNA sequence that hybridizes to the complement of the DNA sequences that encode the amino acid sequence shown in FIG. 4, FIG. 7 or FIG. 8, under highly stringent conditions, e.g hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel F. M. et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & sons, Inc., New York, at p. 2.10.3); and/or (d) any DNA sequence that hybridizes to the complement of the DNA sequences that encode the amino acid sequence shown in FIG. 4, FIG. 7 or FIG. 8, under less stringent conditions, such as moderately stringent conditions, e.g., washing in 0.2×SSC/0.1% SDS at 42° C. (Ausubel et al., 1989, supra), yet which still encodes a functional bg gene product. As used herein, bg gene may also refer to degenerate variants of DNA sequences (a) through (d), including naturally occurring variants. The term "functional bg gene product," as used herein, refers to a gene product encoded by a nucleic acid sequence capable of complementing a recessive, loss-of-function bg mutation.

The invention also includes nucleic acid molecules, preferably DNA molecules, that hybridize to, and are therefore the complements of, the DNA sequences (a) through (d), in the preceding paragraph, and to degenerate variants of the DNA sequences shown in (a) through (d) in the preceding paragraph. Hybridization conditions may be highly stringent or less highly stringent, as described above. In instances wherein the nucleic acid molecules are deoxyoligonucleotides ("oligos"), highly stringent conditions may refer, e.g., to washing in 6×SSC/0.05% sodium pyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos). These nucleic acid molecules may encode or act as bg gene antisense molecules, useful, for example, in bg gene regulation, as antisense primers in amplification reactions of bg gene nucleic acid sequences and/or as hybridization probes for the identification of bg nucleic acid sequences. With respect to diagnostic procedures, such molecules may be used as components of methods whereby, for example, the presence of a particular bg allele responsible for causing a disorder, such as CHS, may be detected.

The invention also encompasses (a) DNA vectors that contain any of the foregoing bg coding sequences and/or their complements (i.e., antisense); (b) DNA expression vectors that contain any of the foregoing bg coding sequences operatively associated with a regulatory element that directs the expression of the coding sequences; and (c) genetically engineered host cells that contain any of the foregoing bg coding sequences operatively associated with a regulatory element that directs the expression of the coding sequences in the host cell. As used herein, regulatory elements include but are not limited to inducible and non-inducible promoters, enhancers, operators and other elements known to those skilled in the art that drive and regulate expression. Such regulatory elements include but are not limited to the cytomegalovirus hCMV immediate early gene, the early or late promoters of SV40 adenovirus, the lac system, the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage A, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase, the promoters of acid phosphatase, and the promoters of the yeast α-mating factors. The invention includes fragments of any of the DNA sequences disclosed herein.

bg gene sequences include, for example, alleles and homologs of genes containing the sequence depicted in FIG. 4, FIG. 7 or FIG. 8, wherein such alleles are present at the same locus as the sequence depicted in FIG. 4, FIG. 7 or FIG. 8 and homologs are genes at other genetic loci within the genome that encode proteins which have extensive homology to one or more domains of the bg gene product. Such bg gene alleles and homologs can be identified and readily isolated, without undue experimentation, by molecular biological techniques well known in the art.

As an example, in order to clone a human bg gene sequence using isolated murine bg gene sequences as disclosed herein, such murine bg gene sequences may be labeled and used to screen a cDNA library constructed from mRNA obtained from appropriate cells or tissues of interest (e.g., a cell or tissue known to express the bg gene in mouse, and/or a cell or tissue known to be affected by CHS in humans, such as, for example, a retinal library). The hybridization washing conditions used should normally be of a lower stringency when the cDNA library is derived from an organism different from the type of organism from which the labeled sequence was derived, but appropriate stringency conditions for the specific sequence and library being utilized will be apparent to those of skill in the art.

Low stringency conditions, for example, are well known to those of skill in the art, and will vary predictably depending on the specific organisms from which the library and the labeled sequences are derived. For guidance regarding such conditions see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press, N.Y.; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y.

Alternatively, the labeled fragment may be used to screen a genomic library derived from the organism of interest, again, using appropriately stringent conditions. Such a screening procedure could be utilized, for example, to identify either bg alleles or bg homolog genes located in different portions of the genome containing sequences encoding one or more domains exhibiting extensive homology to one or more domains encoded by the bg gene.

Further, a bg gene sequence may be isolated from nucleic acid of the organism of interest by performing PCR using two degenerate oligonucleotide primer pools designed on the basis of amino acid sequences within the bg gene product disclosed herein. The template for the reaction may be cDNA obtained by reverse transcription of mRNA prepared from, for example, human or non-human cell lines or tissue known or suspected to express a bg gene allele.

The PCR product may be subcloned and sequenced to ensure that the amplified sequences represent the sequences of a bg gene nucleic acid sequence. The PCR fragment may then be used to isolate a full length cDNA clone by a variety of methods. For example, the amplified fragment may be labeled and used to screen a cDNA library, such as a bacteriophage cDNA library. Alternatively, the labeled fragment may be used to isolate genomic clones via the screening of a genomic library.

PCR technology may also be utilized to isolate full length cDNA sequences. For example, RNA may be isolated, following standard procedures, from an appropriate cellular or tissue source (i.e., one known, or suspected, to express the bg gene, and/or one known to be affected by disorders caused by bg mutations). A reverse transcription reaction may be performed on the RNA using an oligonucleotide primer specific for the most 5' end of the amplified fragment for the priming of first strand synthesis. The resulting RNA/DNA hybrid may then be "tailed" with guanines using a standard terminal transferase reaction, the hybrid may be digested with RNAase H, and second strand synthesis may then be primed with a poly-C primer. Thus, cDNA sequences upstream of the amplified fragment may easily be isolated. For a review of cloning strategies which may be used, see e.g., Sambrook et al., 1989, supra.

bg gene sequences may additionally be used to isolate mutant bg gene alleles. Such mutant alleles may be isolated from individuals either known or proposed to have a genotype which contributes to the symptoms of intracellular vesicle disorders, including CHS. Mutant alleles and mutant allele products may then be utilized in the therapeutic and diagnostic systems described below. Additionally, such bg gene sequences can be used to detect bg gene regulatory (e.g., promoter) defects which can affect intracellular vesicle differentiation and/or function.

A cDNA of a mutant bg gene may be isolated, for example, by using PCR, a technique which is well known to those of skill in the art. In this case, the first cDNA strand may be synthesized by hybridizing an oligo-dT oligonucleotide to mRNA isolated from tissue known or suspected to be expressed in an individual putatively carrying the mutant bg allele, and by extending the new strand with reverse transcriptase. The second strand of the cDNA is then synthesized using an oligonucleotide that hybridizes specifically to the 5' end of the normal gene. Using these two primers, the product is then amplified via PCR, cloned into a suitable vector, and subjected to DNA sequence analysis through methods well known to those of skill in the art. By comparing the DNA sequence of the mutant bg allele to that of the normal bg allele, the mutation(s) responsible for the loss or alteration of function of the mutant bg gene product can be ascertained.

Alternatively, a genomic library can be constructed using DNA obtained from an individual suspected of or known to carry the mutant bg allele, or a cDNA library can be constructed using RNA from a tissue known, or suspected, to express the mutant bg allele. The normal bg gene or any suitable fragment thereof may then be labeled and used as a probe to identify the corresponding mutant bg allele in such libraries. Clones containing the mutant bg gene sequences may then be purified and subjected to sequence analysis according to methods well known to those of skill in the art.

Additionally, an expression library can be constructed utilizing cDNA synthesized from, for example, RNA isolated from a tissue known, or suspected, to express a mutant bg allele in an individual suspected of or known to carry such a mutant allele. In this manner, gene products made by the putatively mutant tissue may be expressed and screened using standard antibody screening techniques in conjunction with antibodies raised against the normal bg gene product, as described, below, in Section 5.3. (For screening techniques, see, for example, Harlow, E. and Lane, eds., 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Press, Cold Spring Harbor.) In cases where a bg mutation results in an expressed gene product with altered function (e.g., as a result of a missense or a frameshift mutation), a polyclonal set of anti-bg gene product antibodies are likely to cross-react with the mutant bg gene product. Library clones detected via their reaction with such labeled antibodies can be purified and subjected to sequence analysis according to methods well known to those of skill in the art.

The Example presented in Section 9, below, demonstrates the successful isolation and sequencing of two bg mutations, each of which causes the production of truncated, non-functional BG proteins.

5.2. PROTEIN PRODUCTS OF THE bg GENE

bg gene products, peptide fragments thereof or fusion proeins, can be prepared for a variety of uses. For example, such gene products, peptide fragments thereof or fusion proteins, can be used for the generation of antibodies, in diagnostic assays, or for the identification of other cellular gene products involved in the differentiation and/or function of intracellular vesicles.

FIG. 4 depicts murine bg gene product amino acid sequence. FIG. 7 depicts the long form, putative full length, human bg gene product amino acid sequence. As shown in FIG. 7, the long form human bg gene product contains 3801 amino acid residues. FIG. 8 depicts the short form human bg gene product encoded by an alternatively spliced short form of bg transcript. As shown in FIG. 8, the human bg gene product encoded by this short form transcript contains 3672 amino acid residues. The bg gene product, sometimes referred to herein as "BG", may additionally include those gene products encoded by the bg gene sequences described in Section 5.1, above.

In addition, bg gene products may include proteins that represent functionally equivalent bg gene products. The term "functionally equivalent bg gene product", as used herein, refers to a gene product encoded by a nucleic acid sequence capable of complementing a bg mutation. Such an equivalent bg gene product may contain deletions, additions or substitutions of amino acid residues within the amino acid sequence encoded by the bg gene sequences described, above, in Section 5.1, but which result in a silent change, thus producing a functionally equivalent bg gene product. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

The bg gene products, peptide fragments thereof or fusion proteins, may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing the bg gene polypeptides, peptides and fusion proteins of the invention by expressing nucleic acid containing bg gene sequences are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing bg gene product coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al., 1989, supra, and Ausubel et al., 1989, supra. Alternatively, RNA capable of encoding bg gene product sequences may be chemically synthesized using, for example, synthesizers. See, for example, the techniques described in "Oligonucleotide Synthesis", 1984, Gait, M. J. ed., IRL Press, Oxford, which is incorporated by reference herein in its entirety.

A variety of host-expression vector systems may be utilized to express the bg gene coding sequences of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, exhibit the bg gene product of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing hg gene product coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the bg gene product coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the bg gene product coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing bg gene product coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the bg gene product being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of bg protein or for raising antibodies to bg protein, for example, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the bg gene product coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The PGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The bg gene coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). Successful insertion of bg gene coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed. (E.g., see Smith et al., 1983, J. Virol. 46: 584; Smith, U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the bg gene coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing bg gene product in infected hosts. (E.g., See Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:3655-3659). Specific initiation signals may also be required for efficient translation of inserted bg gene product coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire bg gene, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a portion of the bg gene coding sequence is inserted, exogenous translational control signals, including, perhaps, the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153:516-544).

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3 and WI38 cell lines.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the bg gene product may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the bg gene product. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that affect the endogenous activity of the bg gene product.

A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adenine phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes can be employed in tk-, hgprt- or aprt- cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler, et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1); and hygro, which confers resistance to hygromycin (Santerre, et al., 1984, Gene 30:147).

Alternatively, any fusion protein may be readily purified by utilizing an antibody specific for the fusion protein being expressed. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA 88: 8972-8976). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the gene's open reading frame is translationally fused to an amino-terminal tag consisting of six histidine residues. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni² +nitriloacetic acid-agarose columns and histidine-tagged proteins are selectively eluted with imidazole-containing buffers.

The bg gene products can also be expressed in transgenic animals. Animals of any species, including, but not limited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, goats, and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate bg transgenic animals.

Any technique known in the art may be used to introduce the bg gene transgene into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to pronuclear microinjection (Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. No. 4,873,191); retrovirus mediated gene transfer into germ lines (Van der Putten et al., 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); gene targeting in embryonic stem cells (Thompson et al., 1989, Cell 56:313-321); electroporation of embryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814); and sperm-mediated gene transfer (Lavitrano et al., 1989, Cell 57:717-723); etc. For a review of such techniques, see Gordon, 1989, Transgenic Animals, Intl. Rev. Cytol. 115:171-229, which is incorporated by reference herein in its entirety.

The present invention provides for transgenic animals that carry the bg transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, i.e., mosaic animals. The transgene may be integrated as a single transgene or in concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko, M. et al., 1992, Proc. Natl. Acad. Sci. USA 89: 6232-6236). The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. When it is desired that the bg gene transgene be integrated into the chromosomal site of the endogenous bg gene, gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous bg gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous bg gene. The transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous bg gene in only that cell type, by following, for example, the teaching of Gu et al. (Gu, et al., 1994, Science 265: 103-106). The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.

Once transgenic animals have been generated, the expression of the recombinant bg gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to assay whether integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include but are not limited to Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and RT-PCR. Samples of bg gene-expressing tissue, may also be evaluated immunocytochemically using antibodies specific for the bg transgene product.

5.3. ANTIBODIES TO bg GENE PRODUCTS

Described herein are methods for the production of antibodies capable of specifically recognizing one or more bg gene product epitopes or epitopes of conserved variants or peptide fragments of the bg gene products.

Such antibodies may include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab')₂ fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. Such antibodies may be used, for example, in the detection of a bg gene product in an biological sample and may, therefore, be utilized as part of a diagnostic or prognostic technique whereby patients may be tested for abnormal levels of bg gene products, and/or for the presence of abnormal forms of the such gene products. Such antibodies may also be utilized in conjunction with, for example, compound screening schemes, as described, below, in Section 5.4.2, for the evaluation of the effect of test compounds on bg gene product levels and/or activity. Additionally, such antibodies can be used in conjunction with the gene therapy techniques described, below, in Section 5.4.3, to, for example, evaluate the normal and/or engineered bg-expressing cells prior to their introduction into the patient.

Anti-bg gene product antibodies may additionally be used as a method for the inhibition of abnormal bg gene product activity, in, for example, instances in which such abnormal activity is due to an increased level of bg gene product or to the presence of mutant, gain-of-function mutant bg gene products. Thus, such antibodies may, therefore, be utilized as part of methods for the treatment of disorders caused by such abnormal bg gene product activity, including, for example, disorders involving abnormal intracellular vesicle differentiation and/or function.

For the production of antibodies against a bg gene product, various host animals may be immunized by injection with a bg gene product, or a portion thereof. Such host animals may include but are not limited to rabbits, mice, and rats, to name but a few. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as a bg gene product, or an antigenic functional derivative thereof. For the production of polyclonal antibodies, host animals such as those described above, may be immunized by injection with bg gene product supplemented with adjuvants as also described above.

Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique of Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production.

In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci., 81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda et al., 1985, Nature, 314:452-454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region.

Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-546) can be adapted to produce single chain antibodies against bg gene products. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.

Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, such fragments include but are not limited to: the F(ab')₂ fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')₂ fragments. Alternatively, Fab expression libraries may be constructed (Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.

5.4. USES OF THE bg GENE, GENE PRODUCTS, AND ANTIBODIES

Described herein are various applications of the bg gene, the bg gene product including peptide fragments thereof, and of antibodies directed against the bg gene product and peptide fragments thereof.

Such applications include, for example, prognostic and diagnostic evaluation of disorders involving abnormal intracellular vesicles, including, for example, abnormal lysosomes, melanosomes, platelet dense granules and cytolytic granules, including, but not limited to Chediak-Higashi syndrome (CHS), and methods for the identification of subjects with a predisposition to such disorders and the identification of individuals carrying mutant bg alleles.

Such methods may, for example, utilize reagents such as the bg gene nucleotide sequences described in Sections 5.1, and antibodies directed against bg gene products, including peptide fragments thereof, as described, above, in Section 5.3. Specifically, such reagents may be used, for example, for: (1) nucleic acid-based techniques for the detection of the presence of bg gene mutations, or the detection of either over- or under-expression of bg gene mRNA relative levels known to be found in the normal state; and (2) peptide-based techniques for the detection of mutant BG proteins or either an over- or an under-abundance of BG relative levels known to be found in the normal state.

The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one specific bg gene nucleic acid or anti-bg gene antibody reagent described herein, which may be conveniently used, e.g., in clinical settings, to diagnose patients exhibiting intracellular vesicle disorder abnormalities.

Nucleic acid-based detection techniques are described, below, in Section 5.4.1. Peptide detection techniques are described, below, in Section 5.4.2.

Additionally, such applications include methods for the treatment of disorders involving abnormal intracellular vesicles, including CHS, as described, below, in Section 5.4.4, and for the identification of compounds which modulate the expression of the bg gene and/or the activity of the bg gene product, as described below, in Section 5.4.3. Such compounds can include, for example, other cellular products which are involved in normal differentiation and/or function of intracellular vesicles.

5.4.1. DETECTION OF bg GENE NUCLEIC ACID MOLECULES

Mutations within the bg gene can be detected by utilizing a number of techniques. Nucleic acid from any nucleated cell can be used as the starting point for such assay techniques, and may be isolated according to standard nucleic acid preparation procedures which are well known to those of skill in the art.

DNA may be used in hybridization or amplification assays of biological samples to detect abnormalities involving bg gene structure, including point mutations, insertions, deletions and chromosomal rearrangements. Such assays may include, but are not limited to, Southern analyses, single stranded conformational polymorphism analyses (SSCP), and PCR analyses.

Such diagnostic methods for the detection of bg gene-specific mutations can involve for example, contacting and incubating nucleic acids including recombinant DNA molecules, cloned genes or degenerate variants thereof, obtained from a sample, e.g., derived from a patient sample or other appropriate cellular source, with one or more labeled nucleic acid reagents including recombinant DNA molecules, cloned genes or degenerate variants thereof, as described in Section 5.1, under conditions favorable for the specific annealing of these reagents to their complementary sequences within the bg gene. Preferably, the lengths of these nucleic acid reagents are at least 15 to 30 nucleotides. After incubation, all non-annealed nucleic acids are removed from the nucleic acid:bg molecule hybrid. The presence of nucleic acids which have hybridized, if any such molecules exist, is then detected. Using such a detection scheme, the nucleic acid from the cell type or tissue of interest can be immobilized, for example, to a solid support such as a membrane, or a plastic surface such as that on a microtiter plate or polystyrene beads. In this case, after incubation, non-annealed, labeled nucleic acid reagents of the type described in Section 5.1 are easily removed. Detection of the remaining, annealed, labeled bg nucleic acid reagents is accomplished using standard techniques well-known to those in the art. The bg gene sequences to which the nucleic acid reagents have annealed can be compared to the annealing pattern expected from a normal bg gene sequence in order to determine whether a bg gene mutation is present.

Alternative diagnostic methods for the detection of bg gene specific nucleic acid molecules, in patient samples or other appropriate cell sources, may involve their amplification, e.g., by PCR (the experimental embodiment set forth in Mullis, K. B., 1987, U.S. Pat. No. 4,683,202), followed by the detection of the amplified molecules using techniques well known to those of skill in the art. The resulting amplified sequences can be compared to those which would be expected if the nucleic acid being amplified contained only normal copies of the bg gene in order to determine whether a bg gene mutation exists.

Additionally, well-known genotyping techniques can be performed to identify individuals carrying bg gene mutations. Such techniques include, for example, the use of restriction fragment length polymorphisms (RFLPs), which involve sequence variations in one of the recognition sites for the specific restriction enzyme used.

Additionally, improved methods for analyzing DNA polymorphisms which can be utilized for the identification of bg gene mutations have been described which capitalize on the presence of variable numbers of short, tandemly repeated DNA sequences between the restriction enzyme sites. For example, Weber (U.S. Pat. No. 5,075,217, which is incorporated herein by reference in its entirety) describes a DNA marker based on length polymorphisms in blocks of (dC-dA)n-(dG-dT)n short tandem repeats. The average separation of (dC-dA)n-(dG-dT)n blocks is estimated to be 30,000-60,000 bp. Markers which are so closely spaced exhibit a high frequency co-inheritance, and are extremely useful in the identification of genetic mutations, such as, for example, mutations within the bg gene, and the diagnosis of diseases and disorders related to bg mutations.

Also, Caskey et al. (U.S. Pat. No. 5,364,759, which is incorporated herein by reference in its entirety) describe a DNA profiling assay for detecting short tri and tetra nucleotide repeat sequences. The process includes extracting the DNA of interest, such as the bg gene, amplifying the extracted DNA, and labelling the repeat sequences to form a genotypic map of the individual's DNA.

The level and/or type of bg gene expression can also be assayed. For example, RNA from a cell type or tissue known, or suspected, to express the bg gene, may be isolated and tested utilizing hybridization or PCR techniques such as are described, above. The isolated cells can be derived from cell culture or from a patient. The analysis of cells taken from culture may be a necessary step in the assessment of cells to be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the bg gene. Such analyses may reveal both quantitative and qualitative aspects of the expression pattern of the bg gene, including activation or inactivation of bg gene expression, as well as reveal the presence or absence of alternatively spliced forms of bg gene transcripts.

In one embodiment of such a detection scheme, a cDNA molecule is synthesized from an RNA molecule of interest (e.g., by reverse transcription of the RNA molecule into cDNA). A sequence within the cDNA is then used as the template for a nucleic acid amplification reaction, such as a PCR amplification reaction, or the like. The nucleic acid reagents used as synthesis initiation reagents (e.g., primers) in the reverse transcription and nucleic acid amplification steps of this method are chosen from among the bg gene nucleic acid reagents described in Section 5.1. The preferred lengths of such nucleic acid reagents are at least 9-30 nucleotides. For detection of the amplified product, the nucleic acid amplification may be performed using radioactively or non-radioactively labeled nucleotides. Alternatively, enough amplified product may be made such that the product may be visualized by standard ethidium bromide staining or by utilizing any other suitable nucleic acid staining method.

Additionally, it is possible to perform such bg gene expression assays "in situ", i.e., directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary. Nucleic acid reagents such as those described in Section 5.1 may be used as probes and/or primers for such in situ procedures (see, for example, Nuovo, G. J., 1992, "PCR In Situ Hybridization: Protocols And Applications", Raven Press, NY).

Alternatively, if a sufficient quantity of the appropriate cells can be obtained, standard Northern analysis can be performed to determine the level of mRNA expression of the bg gene.

5.4.2. DETECTION OF bg GENE PRODUCTS

Antibodies directed against wild type or mutant bg gene products or conserved variants or peptide fragments thereof, which are discussed, above, in Section 5.3, may also be used as intracellular vesicle, including, but not limited to CHS, disorder diagnostics and prognostics, as described herein. Such diagnostic methods, may be used to detect abnormalities in the level of bg gene expression, or abnormalities in the structure and/or temporal, tissue, cellular, or subcellular location of bg gene product. Further, such assays can be utilized to detect the presence or absence of bg gene products encoded by alternatively spliced bg gene transcripts. Given the intracellular vesicles affected by bg mutations, it is possible that the bg gene product is an intracellular gene product. The antibodies and immunoassay methods described below, therefore, have important in vitro applications in assessing the efficacy of treatments for such disorders. Antibodies, or fragments of antibodies, such as those described below, may be used to screen potentially therapeutic compounds in vitro to determine their effects on bg gene expression and bg peptide production. The compounds which have beneficial effects on intracellular vesicle disorders, such as for example, CHS, can be identified, and a therapeutically effective dose determined.

In vitro immunoassays may also be used, for example, to assess the efficacy of cell-based gene therapy for intracellular vesicle disorder, including, for example, CHS. Antibodies directed against bg peptides may be used in vitro to determine the level of bg gene expression achieved in cells genetically engineered to produce bg peptides. Given that the bg gene product may represent an intracellular gene product, such an assessment is, preferably, done using cell lysates or extracts. Such analysis will allow for a determination of the number of transformed cells necessary to achieve therapeutic efficacy in vivo, as well as optimization of the gene replacement protocol.

The tissue or cell type to be analyzed will generally include those which are known, or suspected, to express the bg gene. The protein isolation methods employed herein may, for example, be such as those described in Harlow and Lane (Harlow, E. and Lane, D., 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York), which is incorporated herein by reference in its entirety. The isolated cells can be derived from cell culture or from a patient. The analysis of cell taken from culture may be a necessary step in the assessment of cells to be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the bg gene.

Preferred diagnostic methods for the detection of bg gene products or conserved variants or peptide fragments thereof, may involve, for example, immunoassays wherein the bg gene products or conserved variants or peptide fragments are detected by their interaction with an anti-bg gene product-specific antibody.

For example, antibodies, or fragments of antibodies, such as those described, above, in Section 5.3, useful in the present invention may be used to quantitatively or qualitatively detect the presence of bg gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody (see below, this Section) coupled with light microscopic, flow cytometric, or fluorimetric detection. Such techniques are especially preferred if such bg gene products are expressed on the cell surface.

The antibodies (or fragments thereof) useful in the present invention may, additionally, be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of bg gene products or conserved variants or peptide fragments thereof. In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody of the present invention. The antibody (or fragment) is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of the bg gene product, or conserved variants or peptide fragments, but also its distribution in the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.

Immunoassays for bg gene products or conserved variants or peptide fragments thereof will typically comprise incubating a sample, such as a biological fluid, a tissue extract, freshly harvested cells, or lysates of cells which have been incubated in cell culture, in the presence of a detectably labeled antibody capable of identifying bg gene products or conserved variants or peptide fragments thereof, and detecting the bound antibody by any of a number of techniques well-known in the art.

The biological sample may be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins. The support may then be washed with suitable buffers followed by treatment with the detectably labeled bg gene specific antibody. The solid phase support may then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on solid support may then be detected by conventional means.

By "solid phase support or carrier" is intended any support capable of binding an antigen or an antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.

The binding activity of a given lot of anti-bg gene product antibody may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.

One of the ways in which the bg gene peptide-specific antibody can be detectably labeled is by linking the same to an enzyme and use in an enzyme immunoassay (EIA) (Voller, A., "The Enzyme Linked Immunosorbent Assay (ELISA)", 197, Diagnostic Horizons 2:1-7, Microbiological Associates Quarterly Publication, Walkersville, Md.); Voller, A. et al., 1978, J. Clin. Pathol. 31:507-520; Butler, J. E., 1981, Meth. Enzymol. 73:482-523; Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, Fla.,; Ishikawa, E. et al., (eds.), 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). The enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means. Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The detection can be accomplished by calorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.

Detection may also be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect bg gene peptides through the use of a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein). The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.

It is also possible to label the antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emitting metals such as ¹⁵² Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

The antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in, which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.

5.4.3. SCREENING ASSAYS FOR COMPOUNDS THAT MODULATE bg GENE ACTIVITY

The following assays are designed to identify compounds that bind to bg gene products, bind to other intracellular proteins that interact with a bg gene product, to compounds that interfere with the interaction of the bg gene product with other intracellular proteins and to compounds which modulate the activity of bg gene (i.e., modulate the level of bg gene expression and/or modulate the level of bg gene product activity). Assays may additionally be utilized which identify compounds which bind to bg gene regulatory sequences (e.g., promoter sequences). See e.g., Platt, K. A., 1994, J. Biol. Chem. 269:28558-28562, which is incorporated herein by reference in its entirety, which may modulate the level of bg gene expression. Compounds may include, but are not limited to, small organic molecules which are able to cross the blood-brain barrier, gain entry into an appropriate cell and affect expression of the bg gene or some other gene involved in the pathway or pathways regulating intracellular vesicle differentiation and/or function, or other intracellular proteins. Methods for the identification of such intracellular proteins are described, below, in Section 5.4.3.1. Such intracellular proteins may be involved in the differentiation and/or function of intracellular vesicles, including, but not limited to, lysosomes, melanosomes, platelet dense granules and cytolytic granules. Further, among these compounds are compounds which affect the level of bg gene expression and/or bg gene product activity and which can be used in the therapeutic treatment of disorders involving abnormal intracellular vesicles, including, but not limited to, abnormal lysosomes, melanosomes, platelet dense granules and cytolytic granules, including CHS, as described, below, in Section 5.4.4.

Compounds may include, but are not limited to, peptides such as, for example, soluble peptides, including but not limited to, Ig-tailed fusion peptides, and members of random peptide libraries; (see, e.g., Lam, K. S. et al., 1991, Nature 354:82-84; Houghten, R. et al., 1991, Nature 354:84-86), and combinatorial chemistry-derived molecular library made of D- and/or L- configuration amino acids, phosphopeptides (including, but not limited to members of random or partially degenerate, directed phosphopeptide libraries; see, e.g., Songyang, Z. et al., 1993, Cell 72:767-778), antibodies (including, but not limited to, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab')₂ and FAb expression library fragments, and epitope-binding fragments thereof), and small organic or inorganic molecules.

Compounds identified via assays such as those described herein may be useful, for example, in elaborating the biological function of the bg gene product, and for ameliorating intracellular vesicle disorders such as, for example, CHS. Assays for testing the effectiveness of compounds, identified by, for example, techniques such as those described in Section 5.4.3.1-5.4.3.3, are discussed, below, in Section 5.4.3.4.

5.4.3.1. IN VITRO SCREENING ASSAYS FOR COMPOUNDS THAT BIND TO THE bg GENE PRODUCT

In vitro systems may be designed to identify compounds capable of binding the bg gene products of the invention. Compounds identified may be useful, for example, in modulating the activity of wild type and/or mutant bg gene products, may be useful in elaborating the biological function of the bg gene product, may be utilized in screens for identifying compounds that disrupt normal bg gene product interactions, or may in themselves disrupt such interactions.

The principle of the assays used to identify compounds that bind to the bg gene product involves preparing a reaction mixture of the bg gene product and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex which can be removed and/or detected in the reaction mixture. These assays can be conducted in a variety of ways. For example, one method to conduct such an assay would involve anchoring bg gene product or the test substance onto a solid phase and detecting bg gene product/test compound complexes anchored on the solid phase at the end of the reaction. In one embodiment of such a method, the bg gene product may be anchored onto a solid surface, and the test compound, which is not anchored, may be labeled, either directly or indirectly.

In practice, microtiter plates may conveniently be utilized as the solid phase. The anchored component may be immobilized by non-covalent or covalent attachments. Non-covalent attachment may be accomplished by simply coating the solid surface with a solution of the protein and drying. Alternatively, an immobilized antibody, preferably a monoclonal antibody, specific for the protein to be immobilized may be used to anchor the protein to the solid surface. The surfaces may be prepared in advance and stored.

In order to conduct the assay, the nonimmobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously nonimmobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously nonimmobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the previously nonimmobilized component (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody).

Alternatively, a reaction can be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for bg gene product or the test compound to anchor any complexes formed in solution, and a labeled antibody specific for the other component of the possible complex to detect anchored complexes.

5.4.3.2. ASSAYS FOR INTRACELLULAR PROTEINS THAT INTERACT WITH THE bg GENE PRODUCT

Any method suitable for detecting protein-protein interactions may be employed for identifying bg protein-intracellular protein interactions.

Among the traditional methods which may be employed are co-immunoprecipitation, crosslinking and co-purification through gradients or chromatographic columns. Utilizing procedures such as these allows for the identification of intracellular proteins which interact with ba gene products. Once isolated, such an intracellular protein can be identified and can, in turn, be used, in conjunction with standard techniques, to identify proteins it interacts with. For example, at least a portion of the amino acid sequence of the intracellular protein which interacts with the bg gene product can be ascertained using techniques well known to those of skill in the art, such as via the Edman degradation technique (see, e.g., Creighton, 1983, "Proteins: Structures and Molecular Principles", W. H. Freeman & Co., N.Y., pp.34-49). The amino acid sequence obtained may be used as a guide for the generation of oligonucleotide mixtures that can be used to screen for gene sequences encoding such intracellular proteins. Screening made be accomplished, for example, by standard hybridization or PCR techniques. Techniques for the generation of oligonucleotide mixtures and the screening are well-known. (See, e.g., Ausubel, supra., and PCR Protocols: A Guide to Methods and Applications, 1990, Innis, M. et al., eds. Academic Press, Inc., New York).

Additionally, methods may be employed which result in the simultaneous identification of genes which encode the intracellular protein interacting with the bg protein. These methods include, for example, probing expression libraries with labeled bg protein, using bg protein in a manner similar to the well known technique of antibody probing of λgt11 libraries.

One method which detects protein interactions in vivo, the two-hybrid system, is described in detail for illustration only and not by way of limitation. One version of this system has been described (Chien et al., 1991, Proc. Natl. Acad. Sci. USA, 88:9578-9582) and is commercially available from Clontech (Palo Alto, Calif.).

Briefly, utilizing such a system, plasmids are constructed that encode two hybrid proteins: one consists of the DNA-binding domain of a transcription activator protein fused to the bg gene product and the other consists of the transcription activator protein's activation domain fused to an unknown protein that is encoded by a cDNA which has been recombined into this plasmid as part of a cDNA library. The DNA-binding domain fusion plasmid and the cDNA library are transformed into a strain of the yeast Saccharomvces cerevisiae that contains a reporter gene (e.g., HBS or lacZ) whose regulatory region contains the transcription activator's binding site. Either hybrid protein alone cannot activate transcription of the reporter gene: the DNA-binding domain hybrid cannot because it does not provide activation function and the activation domain hybrid cannot because it cannot localize to the activator's binding sites. Interaction of the two hybrid proteins reconstitutes the functional activator protein and results in expression of the reporter gene, which is detected by an assay for the reporter gene product.

The two-hybrid system or related methodology may be used to screen activation domain libraries for proteins that interact with the "bait" gene product. By way of example, and not by way of limitation, bg gene products may be used as the bait gene product. Total genomic or cDNA sequences are fused to the DNA encoding an activation domain. This library and a plasmid encoding a hybrid of a bait bg gene product fused to the DNA-binding domain are cotransformed into a yeast reporter strain, and the resulting transformants are screened for those that express the reporter gene. For example, and not by way of limitation, a bait bg gene sequence, such as the bg open reading frame sequence in FIG. 4, can be cloned into a vector such that it is translationally fused to the DNA encoding the DNA-binding domain of the GAL4 protein. These colonies are purified and the library plasmids responsible for reporter gene expression are isolated. DNA sequencing is then used to identify the proteins encoded by the library plasmids.

A cDNA library of the cell line from which proteins that interact with bait bg gene product are to be detected can be made using methods routinely practiced in the art. According to the particular system described herein, for example, the cDNA fragments can be inserted into a vector such that they are translationally fused to the transcriptional activation domain of GAL4. This library can be co-transformed along with the bait bg gene-GAL4 fusion plasmid into a yeast strain which contains a lacZ gene driven by a promoter which contains GAL4 activation sequence. A cDNA encoded protein, fused to GAL4 transcriptional activation domain, that interacts with bait bg gene product will reconstitute an active GAL4 protein and thereby drive expression of the HIS3 gene. Colonies which express HIS3 can be detected by their growth on petri dishes containing semi-solid agar based media lacking histidine. The cDNA can then be purified from these strains, and used to produce and isolate the bait bg gene-interacting protein using techniques routinely practiced in the art.

5.4.3.3. ASSAYS FOR COMPOUNDS THAT INTERFERE WITH bg GENE PRODUCT/INTRACELLULAR MACROMOLECULE INTERACTION

The bg gene products of the invention may, in vivo, interact with one or more intracellular macromolecules, such as proteins. Such macromolecules may include, but are not limited to, nucleic acid molecules and those proteins identified via methods such as those described, above, in Section 5.4.3.2. For purposes of this discussion, such intracellular macromolecules are referred to herein as "binding partners". Compounds that disrupt bg binding in this way may be useful in regulating the activity of the bg gene product, especially mutant bg gene products. Such compounds may include, but are not limited to molecules such as peptides, and the like, as described, for example, in Section 5.4.3.1. above, which would be capable of gaining access to the intracellular bg gene product.

The basic principle of the assay systems used to identify compounds that interfere with the interaction between the bg gene product and its intracellular binding partner or partners involves preparing a reaction mixture containing the bg gene product, and the binding partner under conditions and for a time sufficient to allow the two to interact and bind, thus forming a complex. In order to test a compound for inhibitory activity, the reaction mixture is prepared in the presence and absence of the test compound. The test compound may be initially included in the reaction mixture, or may be added at a time subsequent to the addition of bg gene product and its intracellular binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the bg gene protein and the intracellular binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the bg gene protein and the interactive binding partner. Additionally, complex formation within reaction mixtures containing the test compound and normal bg gene protein may also be compared to complex formation within reaction mixtures containing the test compound and a mutant bg gene protein. This comparison may be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal bg gene proteins.

The assay for compounds that interfere with the interaction of the bg gene products and binding partners can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the bg gene product or the binding partner onto a solid phase and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the bg gene products and the binding partners, e.g., by competition, can be identified by conducting the reaction in the presence of the test substance; i.e., by adding the test substance to the reaction mixture prior to or simultaneously with the bg gene protein and interactive intracellular binding partner. Alternatively, test compounds that disrupt preformed complexes, e.g. compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are described briefly below.

In a heterogeneous assay system, either the bg gene product or the interactive intracellular binding partner, is anchored onto a solid surface, while the non-anchored species is labeled, either directly or indirectly. In practice, microtiter plates are conveniently utilized. The anchored species may be immobilized by non-covalent or covalent attachments. Non-covalent attachment may be accomplished simply by coating the solid surface with a solution of the bg gene product or binding partner and drying. Alternatively, an immobilized antibody specific for the species to be anchored may be used to anchor the species to the solid surface. The surfaces may be prepared in advance and stored.

In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds which inhibit complex formation or which disrupt preformed complexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds which inhibit complex or which disrupt preformed complexes can be identified.

In an alternate embodiment of the invention, a homogeneous assay can be used. In this approach, a preformed complex of the bg gene protein and the interactive intracellular binding partner is prepared in which either the bg gene product or its binding partners is labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Pat. No. 4,109,496 by Rubenstein which utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances which disrupt bg gene protein/intracellular binding partner interaction can be identified.

In a particular embodiment, the bg gene product can be prepared for immobilization using recombinant DNA techniques described in Section 5.2. above. For example, the bg coding region can be fused to a glutathione-S-transferase (GST) gene using a fusion vector, such as pGEX-5X-1, in such a manner that its binding activity is maintained in the resulting fusion protein. The interactive intracellular binding partner can be purified and used to raise a monoclonal antibody, using methods routinely practiced in the art and described above, in Section 5.3. This antibody can be labeled with the radioactive isotope ¹²⁵ I, for example, by methods routinely practiced in the art. In a heterogeneous assay, e.g., the GST-bg fusion protein can be anchored to glutathione-agarose beads. The interactive intracellular binding partner can then be added in the presence or absence of the test compound in a manner that allows interaction and binding to occur. At the end of the reaction period, unbound material can be washed away, and the labeled monoclonal antibody can be added to the system and allowed to bind to the complexed components. The interaction between the bg gene protein and the interactive intracellular binding partner can be detected by measuring the amount of radioactivity that remains associated with the glutathione-agarose beads. A successful inhibition of the interaction by the test compound will result in a decrease in measured radioactivity.

Alternatively, the GST-B gene fusion protein and the interactive intracellular binding partner can be mixed together in liquid in the absence of the solid glutathione-agarose beads. The test compound can be added either during or after the species are allowed to interact. This mixture can then be added to the glutathione-agarose beads and unbound material is washed away. Again the extent of inhibition of the bg gene product/binding partner interaction can be detected by adding the labeled antibody and measuring the radioactivity associated with the beads.

In another embodiment of the invention, these same techniques can be employed using peptide fragments that correspond to the binding domains of the bg protein and/or the interactive intracellular or binding partner (in cases where the binding partner is a protein), in place of one or both of the full length proteins. Any number of methods routinely practiced in the art can be used to identify and isolate the binding sites. These methods include, but are not limited to, mutagenesis of the gene encoding one of the proteins and screening for disruption of binding in a co-immunoprecipitation assay. Compensating mutations in the gene encoding the second species in the complex can then be selected. Sequence analysis of the genes encoding the respective proteins will reveal the mutations that correspond to the region of the protein involved in interactive binding. Alternatively, one protein can be anchored to a solid surface using methods described in this Section above, and allowed to interact with and bind to its labeled binding partner, which has been treated with a proteolytic enzyme, such as trypsin. After washing, a short, labeled peptide comprising the binding domain may remain associated with the solid material, which can be isolated and identified by amino acid sequencing. Also, once the gene coding for the intracellular binding partner is obtained, short gene segments can be engineered to express peptide fragments of the protein, which can then be tested for binding activity and purified or synthesized.

For example, and not by way of limitation, a ba gene product can be anchored to a solid material as described, above, in this Section by making a GST-bg fusion protein and allowing it to bind to glutathione agarose beads. The interactive intracellular binding partner can be labeled with a radioactive isotope, such as ³⁵ S, and cleaved with a proteolytic enzyme such as trypsin. Cleavage products can then be added to the anchored GST-bg fusion protein and allowed to bind. After washing away unbound peptides, labeled bound material, representing the intracellular binding partner binding domain, can be eluted, purified, and analyzed for amino acid sequence by well-known methods. Peptides so identified can be produced synthetically or fused to appropriate facilitative proteins using recombinant DNA technology.

5.4.3.4. ASSAYS FOR IDENTIFICATION OF COMPOUNDS THAT AMELIORATE INTRACELLULAR VESICLE DISORDERS

Compounds, including but not limited to binding compounds identified via assay techniques such as those described, above, in Sections 5.4.3.1-5.4.3.3, can be tested for the ability to ameliorate intracellular vesicle disorder symptoms, including symptoms associated with CHS. It should be noted that although bg gene products may be intracellular molecules which are not secreted and have no transmembrane component, the assays described herein can identify compounds which affect bg gene activity by either affecting bg gene expression or by affecting the level of bg gene product activity. For example, compounds may be identified which are involved in another step in the pathway in which the bg gene and/or bg gene product is involved and, by affecting this same pathway may modulate the affect of bg on the development of intracellular vesicle disorders. Such compounds can be used as part of a therapeutic method for the treatment of intracellular vesicle disorders, including, for example, CHS.

Described below are cell-based and animal model-based assays for the identification of compounds exhibiting such an ability to intracellular vesicle disorder symptoms.

First, cell-based systems can be used to identify compounds which may act to ameliorate intracellular vesicle disorder symptoms. Such cell systems can include, for example, recombinant or non-recombinant cells, such as cell lines, which express the bg gene. Further, such cell systems can include, for example, recombinant or non-recombinant cell, such as cell lines, which express mutant forms of the bg gene and/or which exhibit elements of the bg phenotype. For example, bg fibroblast cells, Aleutian mink cells or human Chediak-Higashi cells, as described, below, in Sections 7 and 8, can be used.

In utilizing such cell systems, cells may be exposed to a compound, suspected of exhibiting an ability to ameliorate intracellular vesicle disorder symptoms, at a sufficient concentration and for a time sufficient to elicit such an amelioration in the exposed cells. After exposure, the cells can be assayed to measure alterations in the expression of the bg gene, e.g., by assaying cell lysates for bg MRNA transcripts (e.g., by Northern analysis) or for bg protein expressed in the cell; compounds which increase expression of the bg gene are good candidates as therapeutics. Alternatively, the cells are examined to determine whether one or more aspects of the bg cellular phenotype has been altered to resemble a more normal or more wild type, phenotype, or a phenotype more likely to produce a lower incidence or severity of intracellular disorder symptoms.

In addition, animal-based intracellular vesicle disorder systems, which may include, for example bg mice, may be used to identify compounds capable of ameliorating intracellular vesicle disorder-like symptoms (e.g., bg phenotype). Such animal models may be used as test substrates for the identification of drugs, pharmaceuticals, therapies and interventions which may be effective in treating such disorders. For example, animal models may be exposed to a compound, suspected of exhibiting an ability to ameliorate intracellular vesicle disorder symptoms, at a sufficient concentration and for a time sufficient to elicit such an amelioration of the symptoms in the exposed animals. The response of the animals to the exposure may be monitored by assessing the reversal of disorders associated with intracellular vesicle disorders such as CHS.

With regard to intervention, any treatments which reverse any aspect of the intracellular disorder-like symptoms should be considered as candidates for human intracellular disorder therapeutic intervention. Dosages of test agents may be determined by deriving dose-response curves, as discussed in Section 5.5.1, below.

5.4. COMPOUNDS AND METHODS FOR THE TREATMENT OF INTRACELLULAR VESICLE DISORDERS

Described below are methods and compositions whereby intracellular vesicle disorders, including, but not limited to, CHS may be treated. Loss of normal bg gene product function results in the development of a bg, or intracellular vesicle disorder, phenotype, an increase in bg gene product activity would facilitate progress towards a normal state in individuals exhibiting a deficient level of bg gene expression and/or bg gene product activity.

Alternatively, it is conceivable that symptoms of certain intracellular vesicle disorders may be ameliorated by decreasing the level of bg gene expression and/or bg gene product activity. For example, bg gene sequences may be utilized in conjunction with well-known antisense, gene "knock-out," ribozyme and/or triple helix methods to decrease the level of bg gene expression.

With respect to an increase in the level of normal bg gene expression and/or bg gene product activity, bg gene nucleic acid sequences, described, above, in Section 5.1, can, for example, be utilized for the treatment of intracellular vesicle disorders, including CHS. Such treatment can be administered, for example, in the form of gene replacement therapy. Specifically, one or more copies of a normal bg gene or a portion of the bg gene that directs the production of a bg gene product exhibiting normal bg gene function, may be inserted into the appropriate cells within a patient, using vectors which include, but are not limited to adenovirus, adeno-associated virus, and retrovirus vectors, in addition to other particles that introduce DNA into cells, such as liposomes.

It is conceivable that it may be advantageous to achieve bg gene expression in the brain, given the large number of cell type affected by the bg and CHS phenotypes. As such, gene replacement therapy techniques may be utilized which are capable delivering bg gene sequences to these cell types within patients. Thus, the techniques for delivery of hg gene sequences should be able to readily cross the blood-brain barrier, which are well known to those of skill in the art (see, e.g., PCT application, publication No. WO89/10134, which is incorporated herein by reference in its entirety), or, alternatively, should involve direct administration of such bg gene sequences to the site of the cells in which the bg gene sequences are to be expressed. With respect to delivery which is capable of crossing the blood-brain barrier, viral vectors such as, for example, those described above, are preferable.

Additional methods which may be utilized to increase the overall level of hg gene expression and/or bg gene product activity include the introduction of appropriate bg-expressing cells, preferably autologous cells, into a patient at positions and in numbers which are sufficient to ameliorate the symptoms of intracellular vesicle disorders, including CHS. Such cells may be either recombinant or non-recombinant.

Alternatively, cells, preferably autologous cells, can be engineered to express bg gene sequences which may then be introduced into a patient in positions appropriate for the amelioration of intracellular vesicle disorder symptoms. Alternately, cells which express the bg gene in a wild type in MHC matched individuals, i.e., non-bg individual, and may include, for example, hypothalamic cells. The expression of the bg gene sequences is controlled by the appropriate gene regulatory sequences to allow such expression in the necessary cell types. Such gene regulatory sequences are well known to the skilled artisan. Such cell-based gene therapy techniques are well known to those skilled in the art, see, e.g., Anderson, F., U.S. Pat. No. 5,399,349.

When the cells to be administered are non-autologous cells, they can be administered using well known techniques which prevent a host immune response against the introduced cells from developing. For example, the cells may be introduced in an encapsulated form which, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system.

Additionally, compounds, such as those identified via techniques such as those described, above, in Section 5.4.3, which are capable of modulating bg gene product activity can be administered using standard techniques which are well known to those of skill in the art.

5.5. PHARMACEUTICAL PREPARATIONS AND METHODS OF ADMINISTRATION

The compounds that are determined to affect bg gene expression or gene product activity can be administered to a patient at therapeutically effective doses to treat or ameliorate intracellular vesicle disorders, including CHS. A therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms of intracellular vesicle disorders, including elements associated with the bg phenotype and/or the CHS phenotype.

5.5.1. EFFECTIVE DOSE

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀ /ED₅₀. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

5.5.2. FORMULATIONS AND USE

Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.

Thus, the compounds and their physiologically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration.

For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to give controlled release of the active compound.

For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

6. EXAMPLE Genetic and Physical Mapping of the bg Gene

The Example presented in this Section describes genetic mapping of the murine bg locus into a minimal genetic interval of 0.41 cM+/-0.1 cM on murine chromosome 13. Physical mapping of this minimal bg genetic interval is established herein to be approximately 1 Mb.

6.1 MATERIALS AND METHODS

Mouse crosses segregating beige. Multiple strain crosses were established to maximize inter strain variation in order to facilitate detection of polymorphisms of mapping markers. These included i) (C57BL/6J-bg^(J) X DBA/2J) X C57BL/6J-bg^(J) ; ii) (DBA/2 Co-bg^(8J) X C57BL/6J) X DBA/2 Co- bg^(8J) ; iii) (C3H/HeJ-bg^(2J) X CAST/Ei) X C3H/HeJ-bg^(2j) ; iv) (C57BL/6J-bg^(J) X CAST/Ei) X C57BL/6J-bg^(j) ; v) (DBA/2 Co- bg^(8J) X CAST/Ei) X DBA/2 Co- bg^(8J). The offspring of each of these backcrosses were analyzed, by coat color, for their bg genotype. Genomic DNA was made from a tail clip from each and analyzed for multiple simple sequence length repeat polymorphisms (SSLP). Not all strain combinations were polymorphic for all markers. Additional loci, Nidogen (Nid) and Ras like proteinl (Rasl1) were also genotyped in mice from crosses utilizing CAST/Ei. CAST/Ei vs inbred strain polymorphisms were detected using Single Stranded Conformational polymorphism (SSCP). The primers used for Nid were; forward 5'-CAGTGGAATGACCACCAGGCC-3' (SEQ ID NO:13) and reverse 5'-GTTGCAGGCATGTACCACTAC-3' (SEQ ID NO:14) (from mouse cDNA sequence, NCBI GenInfo ID: 53383) The Rasl1 primers were: forward 5'-TATGAACCTACCAAAGCAGAC-3' (SEQ ID NO:15)and reverse 5'ACTTCGGAAGTAGTTGTCTC (SEQ ID NO:16) (from rat RALA cDNA sequence, GenBank Accession: L19698). The PCR amplification conditions were 94° C. for 2 minutes, 0.15U of AmpliTaq was added for a hot start, followed by 30 cycles of 94° C. for 40 secs, 55° C. for 50 secs, 72° C. for 30 secs. The products were run on either a nondenaturing 8% acrylamide gel at 45W, room temperature for 3 hours, for SSLP analysis or, for SSCP analysis, on a 10% acrylamide gel run at 20W, 4° C. for 2.5 hours. Both types of gel were stained, post running, with SYBR Green I and scanned on an MD Fluorimager.

A linkage map of all loci, including bg was constructed, manually, for proximal MMU Chr 13 by minimizing double and multiple cross overs.

Interspecific backcross mapping. One hundred and eighty eight (C57BL/6J X Mus spretus) X C57BL/6J backcross mice were generated and genomic DNA was prepared to create a BSB mapping panel. A framework map was established using 80 previously mapped SSLP markers which encompassed each chromosome. The conditions used for SSLP analysis were as described above. Linkage maps were constructed using Map manager v2.6.5 (Manley, K. F., 1993, Mammalian Genome 4:303-313).

Additional loci, Nid, Rasl1, Ryanodine receptor 2, (Ryr2) and Neutrophil oxidase factor 2-related sequence, (Ncf2-rs), were also placed on the Chr 13 map using SSCP as each gave a C57BL/6J vs Mus spretus polymorphism. Nid, Rasl, were typed as described above. Ryr2 was analyzed by SSCP using the following primers, (from mouse cDNA sequence, NCBI GenInfo ID: 516278): forward 5'-CAAAGAAAGCCCTCAGAAAC-3' (SEQ ID NO:17)and reverse 5'-AAAGAGGAAAACCCAAGACT-3' (SEQ ID NO:18). Ncf2-rs was also analyzed by SSCP using the following primers (designed from human cDNA sequence, GenBank Accession No. U00776): forward 5'-CAAAAACAAGACACCCAAGT-3' (SEQ ID NO:19)and reverse 5'-TGTGGAATTGAGTGTTGTAG-3' (SEQ ID NO:20).

Physical map of ba minimal interval. The Whitehead mouse YAC library (Research Genetics; Huntsville, Ala.) was screened using SSLP markers including D13Mit173, D13Mit44, D13Mit305 using the PCR conditions described above. The YAC end clones were isolated according to standard methods. The YAC end clones were sequenced on an ABI sequencer. PCR primers from each unique end clone were designed, and used to map the end clone back to the mouse genome on the BSB map to check for chimeric YAC's. Those ends that mapped to the correct region of MMU Chr 13 were subsequently used in further rounds of YAC library screening. Cross addressing of the various SSLP markers and YAC end clones allowed a full YAC contig across the bg minimal genetic interval to be established. BACs were also isolated across the physical region using markers from the region.

In order to size the YACs, yeast genomic DNA was prepared according to the New England Biolabs Imbed procedure. Contour clamped homogeneous electric field (CHEF) electrophoresis was carried out using a CHEF MAPPER electrophoresis apparatus (Bio-Rad Laboratories, Inc., Hercules, Calif.) for 28 hours on a 1% agarose gel with an electric field gradient of 6V/cm at 14° C. and a pulse time of 12.55 sec.

6.2 RESULTS

The mapping procedures described in Section 6.1, above, and depicted in FIG. 1, yielded a genetic map with gave a minimal genetic interval for the bg locus of 0.41±0.1 cM. The data giving this result are summarized herein.

The proximal interval, between bg and D13Mit173, was 2/690 recombinants which was 0.14±0.1 cM. The distal interval, between bg and D13Mit305, was 8/1496 recombinants which was 0.27±0.1 cM. The non-recombinant marker SSLP marker D13Mit44 was typed in 690 animals giving an upper genetic distance (at the 95% confidence limit) between bg and D13Mit44 of 0.4 cM.

The homologous genes Nid, which maps to human Chr 1q43, and Rasl, which maps to human Chr 7p, were also placed on the bg genetic map. Nid was non-recombinant in 690 mice putting it within (at the 95% confidence limit) 0.4 cM of bg. Rasl mapped 0.72 cM distal of bg. The homologous gene Ryr2, which maps to human Chr 1q43-q42, was not mapped in the bg segregating crosses as no polymorphism between any of the strains used was found but it was mapped in the BSB mapping panel. By inference from the BSB map, Ryr2 maps within 1.6 cM of Nid (95% confidence limit). The simplest interpretation of the mapping of homologous genes to the map of proximal mouse Chr 13 around the bg region was that bg is likely to fall within the 1q43 syntenic region on the human genetic map.

The mapping of the locus referred to herein as Ncf2-rs was done using primers designed to amplify human cDNA sequence. Ncf2 has been mapped to human Chr 1cen-q32 and to mouse chromosome 1 (Francke U., 1990, Am. J. Hum. Genet. 47:483-92). The primers designed produced a PCR fragment which mapped to the bg region of Chr13.

The YAC and BAC coverage across the minimal bg genetic interval gives an estimated physical distance of approximately 1 Mb.

7. EXAMPLE YAC Complementation of the Beige Mutation

The experiments presented in this Section describe results which have localized the murine bg gene to a specific interval on murine chromosome 13. Specifically, a complementation-based strategy was utilized to identify two overlapping murine yeast artificial chromosomes (YACs) capable of complementing the murine bg mutation. One of these YACs was tested, via cell fusion studies, and found to be capable of complementing Aleutian mink and human Chediak-Higashi syndrome (CHS) mutant phenotypes, thus strongly suggesting that the mouse, human and mink mutant phenotypes are caused by defects in homologous genes.

7.1. MATERIALS AND METHODS

Isolation and Characterization of YACs. The primers F: 5'-CCAGCCACAGAATACCATCC-3' (SEQ ID NO:21) and R: 5'-GGACATACTCTGCTGCCATC-3' (SEQ ID NO:22) specific for Nidogen (Nid) amino terminal sequences were used to screen the Princeton and Whitehead mouse YAC libraries using the following conditions on an Idaho Technologies Thermal Cycler: 20 sec. 94° hot start, 94° 0 sec./50° 0 sec./72° 15 sec. for 35 cycles. To identify the positive pools, the PCR products were separated on 2% agarose gels, transferred to Nytran membranes and probed by standard hybridization techniques. This screen resulted in the isolation of YACs 195.A8, 151.H1, C9.E7, and C96.G11. YAC 113.G6 was isolated from the Whitehead library using the primers: F:5'-ACCCCAGAACTTGAGAAATAG-3' (SEQ ID NO:23)and R:5'-TGCTGAGGTGATAGGTTTATG-3' (SEQ ID NO:24)specific for the Sequence Tagged Site (STS) 195.A8-right end (R) using the above mentioned PCR conditions. Yeast plugs were prepared according to Gnirke et al. (Gnirke, A. et al., 1993, Genomics 15:659-667). YAC DNA was analyzed by Southern blots or PCR to determine STS and Nid content. The sizes of the YACs were determined using pulsed-field gel electrophoresis on a Bio-Rad CHEF DRII, with a pulse time of 10 to 100 seconds.

YAC End Isolation. One or both end fragments of YACs C9.E7, 195.A8 and 151.H1 were isolated and used to create STS. The end fragments were isolated using inverse PCR according to Joslyn et al. (Joslyn, G., et al., 1991, Cell 66:601-613). Each inverse PCR product was either directly sequenced using the M13-UP and RP sites engineered into the primers, or cloned using Invitrogen's TA cloning kit, and then sequenced using the T7 and SP6 sequencing primers. PCR primers specific for each unique end were created and tested on mouse genomic DNA to determine whether they amplified the expected size product.

YAC End Analysis. Each YAC end was tested to determine whether it was derived from mouse chromosome 13. For YAC end analysis, all STS were tested against a panel of mouse/hamster somatic cell hybrids, some of which harbored mouse chromosome 13 (Kozak, C. A. et al., 1975, Som. Cell Gen. 1:371-382). Each hybrid was tested with the dinucleotide repeat markers D13MIT44 and 173 from Research Genetics specific for the bg/Nid region of mouse chromosome 13 before use to determine whether the relevant region of mouse chromosome 13 was present. Southern blots of these hybrids were then probed with YAC end STS to determine if these markers were present in the mouse chromosome 13 positive hybrids. In some instances, YAC end STS were assayed by PCR. The ends of YAC 195.A8 were further analyzed by genetic mapping onto a panel of interspecific backcrossed beige mice (Jenkins, N. A. et al., 1991, Genomics 9:401-403). Genomic DNA blots of mice from this panel were prepared and hybridized with the single copy STS 195.A8-R and 195.A8-left end (L). The map positions of these two markers was then determined using the program Map Manager v2.6.3 (Manley, K. F., 1993, Mammalian Genome 4:303-313). This analysis placed these two markers on the same genetic interval as bg and Nid.

SpheroDlast fusion and YAC microiniection. All YACs were "retrofitted" with the neomycin resistance gene using the vector pRV1 and homologous recombination in yeast as described by Srivastava and Schlessinger (Srivastava, A. & Schlessinger, D., 1991, Gene 103:53-59). This protocol introduces the neomycin resistance gene and the LYS2 gene into the URA3 gene present in the YAC "right" arm. Spheroplast fusion using retrofitted YACs was performed according to Huxley et al. (Huxley, C. et al., 1991, Genomics 9:742-750) with the following modifications. A 100 ml culture of yeast was grown to an OD₆₀₀ of 3-4 in SD-Lys-Trp. Spheroplasts were prepared using Oxalyticase (Enzogenetics, Oregon) and resuspended in two milliliters of STC (1M Sorbitol, 10 mM CaCl₂, 10 mM Tris pH8). bg mouse fibroblasts (3×10⁶ MCHSF2) were fused to 0.5 ml of the spheroplast preparation in 0.4 mls of 50% PEG/10 mM CaCl₂ (Boehringer Mannheim) for 100, 150 and 200 seconds. The fusion reaction, was diluted with 4.0 mls of serum-free Dulbecco's, incubated at room temperature for twenty minutes, centrifuged at loog and plated into four 100 mm plates. Microinjections were performed according to Gnirke et al. (Gnirke, A. et al., 1993, Genomics 15:659-667). Twenty-four hours after fusion or microinjection, the cells were washed twice with PBS and incubated with Dulbecco's minimal essential media containing 10% FCS and 400-500 μg/ml G418 for three to four weeks. Individual colonies were isolated and expanded to at least 1×10⁶ cells. Genomic DNA was isolated from these colonies using Qiagen Genomic DNA Tips and the DNA used for Southern or PCR analysis. YAC vector sequences which are immediately adjacent to the genomic insert were assayed using PCR primers specific for YAC "left" and "right" arms (Peterson, K. R. et al., 1993, Proc. Natl, Acad. Sci. U.S.A. 90:7593-7597), or by Southern blotting using the YAC vector as a probe. For Southern blotting, ten μg of fibroblast DNA or 2 μg of yeast DNA was cut with HindIII, run on a 0.8% agarose gel, and then transferred to a Nytran membrane. This membrane was then hybridized with the 9.5 kb gel purified HindIII fragment of the retrofitting vector pRV1.

Fluorescent Microscopy. Cells were examined for lysosomal morphology using fluorescent labeling of lysosomes (Perou, C. M. & Kaplan, J., 1993, Som. Cell Mol. Gen. 19:459-468). Briefly, lysosomes were labeled by incubating cells overnight in Dulbecco's plus 10% FCS with 0.5 mg/ml Lucifer Yellow-CH, followed by two washes in culture medium, and a final 2-6 hour chase in medium alone. Lysosomes were visualized on live cells using standard fluorescent microscopy techniques.

Somatic Cell Fusions. Aleutian mink or Human CHS (GM02075A) fibroblast lysosomes were labeled with Lucifer Yellow-CH, while the complemented hg mouse fibroblast colony 195-4 lysosomes was separately labeled with dextran-Texas Red. The two cell populations were trypsinized, mixed together, and fused to one another using UV-inactivated Sendai virus (Perou, C. M. & Kaplan, J., 1993, Som. Cell Mol. Gen. 19:459-468; Schlegel, R. S. & Rechsteiner, M. C., 1975, Cell 5:371-379). The cells were plated and examined twenty-four hours later using fluorescent microscopy. Two photographs of the same field were taken, one to visualize the Lucifer Yellow fluorescence and a second to visualize the dextran-Texas Red. A heterokaryon could be identified by the presence of both dyes within all lysosomes of one cell.

7.2. RESULTS

The experiments reported herein describe, first, the isolation and characterization of murine YACs lying within the physical region in which the bg gene must reside. Further, a complementation-based strategy is utilized to identify which of the isolated YACs were able to complement the bg phenotype, thus significantly narrowing the region within which the bg gene must be located. Third, one of these murine YACs was tested, via cell fusion studies, and found to be capable of complementing the bg phenotype in cells of other species, namely those of Aleutian mink and human.

YAC Characterization. As discussed in Section 6, above, and in Jenkins et al. (Jenkins, N. A. et al., 1991, Genomics 9:401-403) the bg gene is located near Nid on chromosome 13. PCR primers specific for Nid were, therefore, utilized to isolate YACs from the bg/Nid region (FIG. 2A). The Princeton and Whitehead mouse YAC libraries were screened, yielding two YACs from each library. Inverse PCR was used to isolate YAC ends, which were sequenced and used to create STS. Each isolated YAC end was tested to determine if it was derived from mouse chromosome 13. Some of these STS were then used to develop a YAC contig across the bg interval. Of the four Nid positive YACs isolated, 151.H1 and C96.G11 were determined to be unstable and were not used. YACs C9.E7 and 195.A8 were both derived from chromosome 13, mapped to the bg interval, and remained stable with time. A fifth YAC, 113.G6, was isolated from the Whitehead Library using the STS 195.A8-R. It was determined that 113.G6 was chimeric, but had significant overlap (500 kb) with 195.A8. For all subsequent experiments, the retrofitted derivatives of YACs C9.E7, 195.A8, and 113.G6 were used.

Introduction of YACs into bg Mouse Fibroblasts.

Retrofitted YACs were introduced by spheroplast fusion (Huxley, C. et al., 1991, Genomics 9:742-750) or microinjection (Gnirke, A. et al., 1993, Genomics 15:659-667) into fibroblasts derived from a C57BL/6J beige mouse (Perou, C. M. & Kaplan, J., 1993, Som. Cell Mol. Gen. 19:459-468). These cells retained the bg phenotype of abnormally large lysosomes with a clustered perinuclear distribution. This phenotype could be corrected by somatic cell fusion with normal cells (Perou, C. M. & Kaplan, J., 1993, Som. Cell Mol. Gen. 19:459-468). Mutant cells containing YACs were selected for resistance to the neomycin analogue G418, and colonies were examined for lysosomal distribution and morphology by fluorescent microscopy using Lucifer Yellow labeling.

Seven G418 resistant colonies from several independent spheroplast fusions using YAC 195.A8 were obtained. The efficacy of YAC transfer using spheroplast fusion was extremely low as determined by G418-resistance. A frequency of colony formation of 10⁷ was calculated. Southern and PCR analyses (Peterson, K. R. et al., 1993, Proc. Natl, Acad. Sci. U.S.A. 90:7593-7597) confirmed that all resistant colonies contained YAC "right" arm vector sequences. Only three of the seven colonies, however, contained YAC "left" arm vector sequences, indicating that the other four colonies contained only a fragment of the YAC. Of the seven colonies, five showed a complemented phenotype (FIG. 3B). These five colonies included the three complete YAC colonies and two of the fragmented YACs. Complemented cells showed dramatically smaller lysosomes than the parental bg cells (FIGS. 3A and 3C). Other features indicating a corrected phenotype included lysosomes which were no longer clustered in the perinuclear region, and the disappearance of tubular lysosomes. Tubular lysosomes are frequently seen in macrophages but are not observed in normal fibroblasts. Tubular lysosomes are seen, however, in bg mouse and Aleutian mink fibroblasts.

YAC 113.G6 was introduced into murine bg fibroblasts using spheroplast fusion and two independent colonies were obtained. One colony was complemented and contained sequences from both YAC arms. The other colony was not complemented and contained only a fragmented copy of the YAC. YAC C9.E7 was microinjected into the bg mouse cell line and thirty independent colonies were obtained, five of which contained both YAC vector arms as determined by PCR. All of these colonies retained the bg phenotype. Cells resistant to G418 due to YAC introduction, either uncomplemented cells carrying fragmented YACs or C9.E7 microinjected cells, showed no complemented features regardless of the concentration of G418 employed.

In the complemented colonies isolated using a G418 concentration of 400-500 μg/ml, it was observed that not all cells showed the complemented phenotype. Some colonies appeared to contain a mixture of bg and complemented cells (FIG. 3B). Two possibilities were considered. First, the colonies, as removed from the plate, might contain a mixture of G418-resistant complemented cells and G418-resistant bg cells. Second, the YAC might be unstable and, at the concentration of G418 employed, some cells may lose the YAC and revert to the bg phenotype. To distinguish between these possibilities, the cell line 195-4 was incubated in 800 μg/ml G418 and lysosomal morphology examined after 10 days. Examination of multiple fields and several hundred cells revealed very few (<1%) bg appearing cells (FIG. 3C). When these complemented cells were incubated in the absence of G418 there was a time-dependent return to the bg phenotype. Seven days after the removal of G418, approximately 1.0% of the cells showed the mutant phenotype, but after thirty days greater than 30% of the cells showed the bg phenotype (FIG. 3D). These results demonstrate that complementation of the mutant phenotype was YAC-dependent.

The fact that not all YACs complement, and that not all spheroplast fusion generated colonies were complemented, suggests that the act of introducing these YACs along with yeast DNA does not cause the reversion of the bg phenotype. Further, the fact that only certain fragmented YAC 195.A8 or 113.G6 YAC molecules failed to correct the phenotype, suggests that fragmented YACs can be utilized as part of a strategy to localize the relevant gene.

Complementation of the CHS Defect in Other Species by a Murine YAC. To analyze the nature of defective genes in different species exhibiting bg phenotypes, both complemented and uncomplemented 195.A8 YAC containing bg cells were fused with cultured Aleutian mink or human CHS derived cells (Perou, C. M. & Kaplan, J., 1993, Som. Cell Mol. Gen. 19:459-4682). When Aleutian mink cells were the recipient cell line, complementation occurred only when the complemented bg mouse cell lines were used. Identical results were obtained with the human CHS fibroblast cell line GM02075A. It was necessary to fuse complemented murine cells to mink and human cells as neither mink nor human cells will accept YACs using the spheroplast fusion or microinjection protocols. These results strongly support the hypothesis that a similar gene (or genes) is responsible for the Chediak phenotype.

It was found that the choice of cell lines was the most important parameter in determining the efficacy of YAC transfer. No G418-resistant colonies were ever obtained using primary human, mouse, or mink fibroblasts as the recipients. Colonies were obtained using the long term cultured bg mouse cell line MCHSF2. A ten to twenty fold increase in the frequency of transformants was obtained using mouse L-cells. These results suggest that increased chromosome instability resulting from long term culture may contribute to increased transformation efficiency.

8. EXAMPLE: Positional Cloning of a Candidate Beige Gene

The Example presented in this Section describes the cloning of a gene, referred to here as the 22B/30B gene, which represents a candidate murine beige (bg) gene. Extending the studies described in Sections 6 and 7, above, the 22B/30B gene was identified via a refinement of the YAC mapping data presented above, couple with a positional cloning strategy. Characterization of the 22B/30B gene indicated that the gene produces an approximately 12-14 kb mRNA that encodes a novel protein exhibiting strong nucleotide homology to multiple expressed sequence tags (ESTs), including human ESTS.

8.1. MATERIALS AND METHODS

YAC Characterization. The Princeton and Whitehead mouse YAC libraries were screened by PCR with primers specific for the right end sequence of YAC 195.A8, as described above. This screen resulted in the isolation of two additional YACS, 137.A10 and B27.F7. An additional YAC from this region was isolated from the Princeton library using primers from the end sequences of the cDNA clone 22B, described in Section 8, below. The F primer was 5'-ATTGGCTAGTGTGTGCAGAC-3' (SEQ ID NO:25)and the R primer was 5'-GAAGCAGATGACTGAGCAGA-3' (SEQ ID NO:26). PCR reactions were performed on an Idaho Technologies Thermal Cycler under the following conditions: 20 sec. 94° C. hot start, 94° C. 0 sec./55° C. 0 sec./72° C. 30 sec. for 30 cycles.

All other YAC techniques were as described, above, in Section 7.1.

Isolation of cDNAs and Preparation of Plugs. Agarose blocks containing yeast chromosomal and YAC 195.A8 DNA were prepared as described in Gnirke et al (Gnirke, A. et al., 1993, Genomics 15:659-667), loaded in a 1%, 0.5×TBE gel and electrophoresed in a Bio-Rad DRII clamped homogeneous electric field (CHEF) apparatus (Bio-Rad Laboratories, Inc., Hercules, Calif.) at 200 V with a constant pulse time of 60 sec. for 24 hrs. The YAC was excised and purified using the GeneClean II Kit according to manufacturers instructions (Bio 101, Inc., La Jolla, Calif.). Gel-purified YAC DNA was radiolabelled with ³² P-dCTP by random priming. The hybridization probe was pre-competed with 100 μg of sonicated genomic mouse DNA, 50 μg of mouse COT-1 DNA (GIBCO BRL, Gaithersburg, Md.) and 20 μg of sonicated pYAC55 DNA (Sigma, St. Louis, Mo.) for 2 hrs at 65° C. Filters containing plaques from a C57BL/6J mouse E16.5 cDNA library (Stratagene, La Jolla, Calif.) were prehybridized at 65° C. for 6-8 hrs in RapidHyb buffer (Amersham, Arlington Heights, Ill.) containing 100 μg/ml sonicated mouse genomic DNA, 4 μg/ml COT-1 DNA and 2 μg/ml sonicated pYAC55 DNA. Hybridization proceeded overnight at 65° C. Filters were washed to 0.1×SSC at 65° C. Clones positive after a secondary screen were recovered as phagemids.

Genomic DNA Isolation and Southern Blots. High molecular weight mouse DNA for Southern Blots and PCR analysis was either purchased from the Jackson Labs (Bar Harbour, Me.) or isolated using a Qiagen tip 2500 (Qiagen, Inc., Chatsworth, Calif.). Southern blots were prepared and hybridized according to (Jenkins, N. A. et al., 1982, J. Virol. 43:26-36), exposed to Fuji Imaging Plates, Type BAS-IIIS and visualized using a Fujix Bas 1000 Phosphoimager (Fuji Film I & I, Fuji Medical Systems U.S.A., Inc., Stamford, Conn.).

RNA Isolation, Northern Blots. Total RNA was isolated from various mouse tissues and cultured mouse and human melanoma cells using the RNA STAT-60 reagent (Tel-Test "B", Inc., Friendswood, Tex.) according to manufacturer's instructions. For Northern blot preparations, 25 μg of this RNA was run on a 1.5% denaturing gel and transferred overnight onto Zeta pore membrane (CUNO, Inc., Meriden, Conn.) in 10×SSC. Filters were hybridized with a gel purified 811 bp HindIII +Pst I fragment from the clone 30B that was radiolabeled with ³² P-dCTP by random priming. Hybridization was performed at 65° C. overnight in QuikHybe Hybridization Solution (Stratagene, La Jolla, Calif.). Filters were washed to 0.1×SSC at 65° C. and visualized by X-ray film autoradiography.

8.2. RESULTS

YAC characterization. The minimal bg interval was refined by further in vitro complementation of bg murine fibroblasts with additional YACS (FIG. 2B). First, it was demonstrated that YAC151.H1, which contains restriction fragments in common with YAC195.A8, as defined by fingerprinting with COT-1 DNA, was not capable of complementing bg. Furthermore, YAC137.A10 which is nearly identical to that of YAC 113.G6, also failed to complement the bg phenotype. These studies, therefore, demonstrate that the minimal bg region must lie between the proximal end of YAC137.A10 and the distal end of YAC151.H1.

Isolation of candidate genes in the bg minimal region. The complementing YAC195.A8 (See Section 7, above) was gel purified, radiolabelled and used to isolate clones from an E16.5 day mouse embryo cDNA library. Forty five clones were isolated. Based on sequence analysis and mapping to the YAC-defined physical map, six genes were defined.

Of particular note was a gene, referred to herein as the 22B/30B gene, defined by two cDNA clones, 30B and 22B. These clones had 447 bp of overlap sequence, with 30B extending more 5' than 22B, and were located within the region predicted to contain the bg gene. In order to determine whether the cDNA clones 22B and 30B mapped physically to the interval predicted to contain the bg gene, the clones were used as probes against restriction enzyme digested YAC DNA. The non-complementing YAC137.A10 lacked two HindIII 30B hybridizing bands that were present in complementing YAC113.G8. Likewise, YAC151H.1 lacked some HindIII bands hybridizing with 22B.

Based upon the complementation data, it was predicted that the complete bg gene would lie in the region of overlap between YACs 113.G6 and 195.A8, but would be disrupted or absent from the non-complementing YACs 137.A10 and 151.H1. This was the pattern observed for the 22B/30B gene, making it a candidate for the bg gene.

Sequence of 22B/30B gene. Sequencing of the two overlapping cDNA clones, 22B and 30B, of the putative bg gene totaled 6831 bp of contiguous sequence (FIG. 4; SEQ ID NO:1). 6559 bp of this was open reading frame followed by a stop codon at nucleotide 6560 and 269 bp of the 3' untranslated region (with 30B present 5' of this contiguous sequence relative to 22B).

The 22B/30B protein sequence predicted from the 22B/30B nucleotide sequence is 2186 amino acids and encodes a novel protein (FIG. 4; SEQ ID NO:2). A BLASTX (1993, Nature Genetics 3:266-272) search with the 22B/30B protein amino acid sequence did, however, identify significant homologies to several sequences. Such sequences included an anonymous S.cerievisiae protein, YCRO32w, encoded by a 7 kb MRNA, two C. elegans novel proteins, T01H10.8 and F10F2.1 and a human gene, cell division control protein 4-related protein, CDC4L. Amino acid residues 1520-1807 of the 22B/30B protein sequence exhibited the highest level of amino acid conservation. Within this region, the S.cerievisiae and C. elegans proteins showed 50% identity and 75% similarity to murine 22B/30B. The homology to the human CDC4L protein spanned a shorter segment (22B/30B amino acid residues 1675-1806), but again showed 50% identity.

A known protein motif was found within the 22B/30B amino acid sequence. Specifically, a WD40 or G protein-beta subunit repeat motif (van der Voorn, L. & Ploegh, H. L., 1992, FEBS Lett. 307:131-134) was found to be located at amino acid residue 2016-2030. This motif was originally identified in the β-subunit of the G-protein transducin (Duronio, R. J. et a., 1992, Proteins 13:41-56), and is thought to be involved in mediating protein-protein interactions (Wang, D. S. et al., 1994, Biochem. Biophys. Res. Comm. 203:29-35). None of the proteins found to be homologous to the 22B/30B protein sequence contain such a motif.

Comparison of the 22B/30B DNA sequence to the dbEST database identified homologies to ESTs from two human cDNA libraries. Specifically, 22B/30B nucleotides 725-942 were 82% identical to human cDNA clone H51623 isolated from a fetal liver and spleen cDNA library, 22B/30B nucleotides 1530-1596 were 88% identical and 22B/30B nucleotides 1596-1842 were 74% identical to the human cDNA clone H50968 isolated from the same fetal cDNA library. 22B/30B nucleotides 1096-1269 were 89% identical to the cDNA clone Z21358 isolated from an adult human testis library. 22B/30B nucleotides 1092-1164 were 87% identical and nucleotides 1165-1302 were 91% identical to the human cDNA clone Z21296 isolated from the same testis cDNA library. In summary, the 22B/30B sequence from approximately nucleotide 725 to nucleotide 1842 appeared to be highly homologous at the nucleotide level to one or more human gene sequences.

Expression of the 22B/30B gene. PCR analysis from reverse transcribed murine mRNA was used for detecting expression of the 22B/30B gene. Such an analysis indicated that the 22B/30B message was expressed in each of the tissues tested, namely liver, spleen, kidney, thymus, muscle, fat, heart, lung, stomach, pancreas and cultured fibroblasts. Using an 811 bp probe from the most 5' end of the available cDNA 22B/30B sequence, a Northern blot of mRNA from two human melanoma cell lines, WM-115 and WM266-4 and from the mouse B16 melanoma cell line showed hybridization to an approximately 12-14 kb message. It should be noted that the 811 bp probe used overlapped with the portion of the 22B/30B sequence discussed above that exhibits 82% identity to a human EST.

9. EXAMPLE Identification of the Beige Gene Via Beige Mutation Detection

The Example presented herein describes the successful identification of the bg gene, the homolog of the gene responsible for the human Chediak-Higashi syndrome (CHS), via the sequencing of two independent mutant bg alleles. The mutation detection analysis revealed that the bg gene corresponds to the 22B/30B gene described in Section 8, above.

9.1. MATERIALS AND METHODS

Southern blot/Genomic DNA isolation. The procedures utilized were as described in Section 8.1, above.

RT-PCR. RNA was isolated as described, above, in Section 8.1. Reverse transcription-polymerase chain reactions (RT-PCR) were carried out as follows: briefly, 0.5 μg of total RNA was reverse transcribed into cDNA using equal concentrations of random and oligo(dT)₁₅ primers and AMV reverse transcriptase (Promega Corp., Madison, Wis.) in a final volume of 200 μl. One μl of each reaction was amplified with 0.25 μM of each of the appropriate primers. The primers were as follows: 22B-5F--5'-TCTTCTTGTCCTGCCTGATGCT-3' (SEQ ID NO:27); 22B-D11--5'-GTGCTTCACTTCCTCCAGATC-3' (SEQ ID NO:28); 22B-D6--5'-GCCTCATTCCAGCGAAGC-3' (SEQ ID NO:29); 22B-D10--5'-CTGGATAGCAGGTGATGGGTGGTTA-3' (SEQ ID NO:30). Amplifications were carried out in a final volume of 25 μl in 1×PCR buffer containing 1.5 mM MgCl₂, 0.5 Units Ampli Taq polymerase (Perkin-Elmer-Cetus). After an initial denaturation step at 94° C. for 2 mins, samples were subjected to 35 cycles of 40 sec at 94° C., 50 sec at 57° C., and 2 mins at 72° C. Following a 10 mins final extension at 72° C., samples were stored at 4° C. PCR products were separated by electrophoresis through 2%, 1×TBE agarose gels.

PCR: Mouse genomic DNA (C57BL/6J, C57BL/6J-ba/ba, Satin/Beige-bg/bg DNA) was amplified using the following primers: 228F: 5'-TGCTGTGGATTATATGAACTC-3' (SEQ ID NO:31)and 228R: 5'-GGTCTCTATTAGTCCGAGAAC-3' (SEQ ID NO:32). Amplification parameters were as follows: 2 minutes hot start 94° C., 94° C. 30 seconds/52° C. 30 seconds/72° C. 4 minutes, for 30 cycles on a Perkin-Elmer DNA Thermal Cycler.

9.2 RESULTS

bg gene mutation detection. Described herein are bg gene mutation studies which reveal that the gene corresponding to the 22B/30B gene, described above, in Section 8, corresponds to the murine bg gene. Specifically, nucleotide defects within two bg mutant alleles are demonstrated to lie within the 22B/30B region and to result in the production of C terminally truncated proteins.

The original bg mutation arose in a radiation experiment at the Oak Ridge National Laboratory. Hence it was probably radiation induced and was either on a chromosome originating from the C3H/R1 or the 101/R1 inbred strains of mice. Because the original bg mutation was radiation-induced, it was possible that the mutation could be visible via Southern blot analysis. There have been many subsequent re-mutations of the mouse bg gene, all of which have arisen spontaneously. Some of these are extinct with no surviving tissues or DNA. For others, for example C57BL-bg^(10J) and C57BL-bg^(11J) although the mutation is extinct there is DNA available (Jackson Laboratories), and for others, the mutation is still available as a live mutant stock, e.g. SJL-bg, C57BL-bg^(J), C3H/HeJ-bg^(2J), and DBA/2J-Co-bg^(8J). Southern blot analysis of these multiple bg alleles and their appropriate normal controls showed no polymorphic bands for probes from either the 5' or 3' regions of the 22B/30B gene sequence, although a probe, 22B/30B nucleotides 6489-6719, did make it possible to determine that the original bg allele arose on a C3H-like chromosome, not a 101/R1 derived chromosome. In contrast, when a 510 bp fragment, 22B/30B nucleotides 1618-2127, was used as a probe, the original mutant bg allele showed altered bands for 7 out of 9 enzymes (FIGS. 5A-5D).

PCR primers, as described above, in Section 9.1, were designed which surrounded and spanned the 510 bp region and were used to amplify genomic DNA and cDNA. One primer set, designated 228F and R, amplified a 2 kb genomic fragment from C57BL/6J but amplified a 3 kb genomic fragment from the strain carrying the original bg allele. A similar set of primers, 22B-5F and 22B-11, was used to amplify cDNA prepared from the kidneys of C57BL/6J, C3H/HeJ and SJL/J-bg. A single band of 312 bp was detected in both C57BL/6J and C3H/HeJ. cDNA from the SJL/J-bg, mouse, however, produced two bands, 428 bp and 637 bp.

Both the RT-PCR products, as well as the genomic DNA PCR products were isolated and directly sequenced using standard procedures. Sequencing of the amplified products revealed that the bg mutation was located within the 22B/30B gene. Specifically, analysis of the amplified sequences revealed that the increased size of the genomic product from the bg allele was the result of an incomplete LINE 1 element (Burton, F. H. et al., 1986, J. Mol. Biol. 187:291-304) insertion into an intron contained within the 30B/22B gene's genomic DNA. As this element contained adventitious splice donor and acceptor sites, two aberrant mRNAs were created that each result in a frame shift. The 428 bp RT-PCR product had a 116 bp LINE 1 insertion between 22B/30B nucleotides 2235 and 2236, while analysis of the larger product demonstrated a 325 bp LINE 1 insertion at this same location. Both of these two LINE 1 insertions results in the introduction of stop codons and in a 22B/30B protein product that is truncated by 1442 amino acids. See FIG. 6 for a diagram depicting the location of these insertions.

Analysis of another bg allele, bg^(8J), by sequencing of an RT-PCR product produced using primers 22B-D6 and 22B-D10, identified a C to T base change creating a stop codon at 22B/30B bp 2027. The mutation resulted in the production of a truncated 22B/30B protein missing the last 1511 amino acids.

It should be noted that the truncated proteins produced by each of the bg (22B/30B) mutant alleles lack the amino acid sequence homologous to S. cerevisiae, C. elegans and human CDC4L genes and also delete the putative WD40 motif (described, above, in Section 8).

In summary, two independent bg gene mutations were revealed to lie within the sequence of the 22B/30B gene, thus presenting compelling evidence identifying the 22B/30B gene to, in fact, correspond the bg gene.

10. EXAMPLE Identification and Characterization of the Humane bg Gene

The Example presented herein describes the successful identification and characterization of cDNA molecules corresponding to the human beige (bg) gene. Characterization of the isolated cDNA molecules revealed that the human bg gene undergoes alternative splicing, yielding long (putative full length) and short forms of bg transcripts and bg gene products, as described below.

10.1. MATERIALS AND METHODS

cDNA cloning. A human retina λgt10 library (Cat. No. HL1132a; Clontech, Palo Alto Calif.) was screened with a mixture of three DNA fragments isolated from mouse beige clones. They were, in order from 5' to 3', 30B (bp 82-921 of FIG. 4), 22B (bp 1650-2160 of FIG. 4), and K2+K5 (bp 6520-6750 of FIG. 4).

The three probes were labeled with ³² P by random priming and hybridized with filters representing 10⁶ clones overnight at 42° C. in Church's buffer (7% SDS, 250 mM NaHPO₄, 2 μM EDTA, 1% BSA). The filters were washed in 2×SSC, 1% SDS at 42° C. Positive plaques were replated and treated in the same manner. Phage DNA was prepped by a standard plate lysate method. After digestion of the phage DNA with EcoRI, cDNA inserts were isolated and subcloned into pBluescript (Stratagene; La Jolla Calif.) for DNA sequencing. DNA sequencing was performed according to standard techniques.

cDNA identified in the above screening was used to probe a λgtlo human fetal liver library (Cat. No. HL3020a; Clontech, Palo Alto Calif.) and the human retina library described above. Filters representing 10⁶ clones of each library were hybridized at 65° C. overnight with ³² P labelled probe in Church's buffer and washed in 0.1×SSC, 0.1% SDS at 65° C. Positive plaques were replated and rescreened in the same manner. Phage DNA was prepared, and cDNA inserts were isolated and subcloned as described above.

10.2. RESULTS

In order to identify the human bg gene, murine bg gene sequences were used to screen human cDNA libraries. Screening, phage isolation and details are presented in Section 10.1, above.

First, a murine bg sequences were used to probe a human retina cDNA library. This screen resulted in the identification of a phage containing a 2 kb cDNA insert (designated fvhx004). The cDNA insert was isolated and subcloned. The fvhx004 cDNA insert was then used to rescreen the human retina cDNA library and to screen a λgt10 human fetal liver library, as described in Section 10.1, above. This screen yielded two positive phage from the human fetal liver library. One phage contained a 4.4 kb cDNA insert (designated fvh1006) and the second contained a 6.3 kb cDNA insert (designated fvh1009). A 3 kb subclone of fvh1006 which overlapped the fvh1009 clone was designated fvh1006a. Additional subclones of fvh1006 were designated fvh1006b (a 1 kb sublcone) and fvh1006c (a 400 bp subclone). An additional positive phage was also isolated from the human retina library. This phage contained a 2 kb cDNA insert (designated fvhx003a). A 1.1 kb HindIII/EcoRV fragment from fvh1009 was used to rescreen the human retina library. This screen resulted in one positive phage containing a 1.6 kb insert, designated fvhx015.

The isolated clones were sequenced according to standard procedures. A database search using human bg nucleotide sequence revealed extensive homology to human cDNA clones H51623 (96% identity), Z21358 (99% identify) and Z21296 (97% identity), as indicated in parentheses. These clones were described in Section 9, above.

Comparison of the human bg sequence with that of mouse bg sequences revealed a 378 base pair region present in mouse sequence which was absent from the sequence obtained from the isolated human clones. PCR of both the retina and liver libraries with primers flanking this sequence, however, revealed that the sequence was present in both these libraries. Sequencing of the resultant PCR products, coupled with the sequence obtained from the isolated clones, produced what is referred to below as the "long" form of bg gene sequence, while the sequence of the isolated clones, alone, yielded what is referred to below as the "short" form of bg gene sequence.

FIG. 7 presents the long form (putative full length) human hg gene nucleic acid sequence. FIG. 7 further depicts the derived amino acid sequence encoded by the long form (putative full length) human bg gene nucleic acid sequence shown therein. As shown in FIG. 7, the predicted long form human bg gene product contains 3801 amino acid residues. As in the mouse bg gene product described in Section 8, above, the human bg gene product contains a WD40 or G protein-beta subunit repeat motif. In the long form human bg gene product this motif is present at amino acid residues 3694-4708.

FIG. 8 presents the short form human bg gene nucleic acid sequence. FIG. 8 further depicts the derived amino acid sequence encoded by the short form human bg gene nucleic acid sequence shown therein. As shown in FIG. 8, the predicted short form human bg gene product contains 3672 amino acid residues. It is missing base pairs 7544-7921 of the long form depicted in FIG. 7. The short form bg nucleic acid sequence retains the same frame as the long form throughout its length and encodes a bg gene product which is missing amino acid residues 2451-2577 of the long form depicted in FIG. 7. The WD40 sequence motif is present in the short form bg gene product at amino acid residues 3565-3579 depicted in FIG. 8.

The present invention is not to be limited in scope by the specific embodiments described herein, which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

    __________________________________________________________________________     #             SEQUENCE LISTING     - (1) GENERAL INFORMATION:     -    (iii) NUMBER OF SEQUENCES: 32     - (2) INFORMATION FOR SEQ ID NO:1:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 6830 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (ix) FEATURE:               (A) NAME/KEY: Coding Se - #quence               (B) LOCATION: 1...6558               (D) OTHER INFORMATION:     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:     #GGG CAG AGG AAG ACT GAT      48GG GTC TCT     #Gly Gln Arg Lys Thr Asp Ile Trp Val Ser     #                 15     #ACA AGC TTA TCA TCA GAC      96CA AGA AAA     #Thr Ser Leu Ser Ser Asp Leu Pro Arg Lys     #             30     #TGC TTA ACA TCC CAA GAA     144TT GGT CAT     #Cys Leu Thr Ser Gln Glu Met Ile Gly His     #         45     #CTG GGG AAC TTG CTC CTC     192AA TGG GAC     #Leu Gly Asn Leu Leu Leu Gly Lys Trp Asp     #     60     #GCC TTT TTC CTG TAT GCT     240CA CAA GAG     #Ala Phe Phe Leu Tyr Ala Gly Ser Gln Glu     # 80     #TGT AAA TAT GGA CAG CCA     288TC ATG CCG     #Cys Lys Tyr Gly Gln Pro Ser Ile Met Pro     #                 95     #GAC ATT TTG AGA TGT GAT     336TT AAT AAA     #Asp Ile Leu Arg Cys Asp Tyr Ile Asn Lys     #            110     #GAA GTG GAT GTT GGT CTC     384CC AAG AAA     #Glu Val Asp Val Gly Leu Met Thr Lys Lys     #        125     #ACT TGC TGT CCT GCT CAG     432TT TAT ACA     #Thr Cys Cys Pro Ala Gln Val Val Tyr Thr     #    140     #AAG GGC CAA GTG AAA ACT     480TT CGA CTC     #Lys Gly Gln Val Lys Thr Val Ile Arg Leu     #160     #GAA GCC CAG AGC ATC TTG     528GC TCA AAG     #Glu Ala Gln Ser Ile Leu Phe Ser Ser Lys     #                175     #CCT ACG GAA TGT AAA GCC     576GC CTC CAA     #Pro Thr Glu Cys Lys Ala Lys Gly Leu Gln     #            190     #GCT GGC ACA TTT GTT TTT     624TT GGT GGG     #Ala Gly Thr Phe Val Phe Glu Ile Gly Gly     #        205     #TGT GAA GAA ACT CAA GCA     672TT AGT AGC     #Cys Glu Glu Thr Gln Ala Glu Leu Ser Ser     #    220     #AAG TAC AGC CAA CAG AGA     720CT TTA ATT     #Lys Tyr Ser Gln Gln Arg Leu Ser Leu Ile     #240     #TCT ATG ATT CAC CAA GTG     768AT GGA CTC     #Ser Met Ile His Gln Val Cys Asn Gly Leu     #                255     #CAC ATT TTG AAG ACC CTT     816TT GGC TTT     #His Ile Leu Lys Thr Leu Ile Val Gly Phe     #            270     #CAC GTC AGT GAG CAT GGA     864AA GTT ATC     #His Val Ser Glu His Gly Glu Glu Val Ile     #        285     #ATA ATC CAA GAT GTT AAG     912CT CAT GCT     #Ile Ile Gln Asp Val Lys Glu Ser His Ala     #    300     #ATA TGG AAT AAG GCA GAG     960AC TGG AAG     #Ile Trp Asn Lys Ala Glu Leu Asp Trp Lys     #320     #TTG GAA GTC CTC ATC CGG    1008TA GCA GCT     #Leu Glu Val Leu Ile Arg Leu Leu Ala Ala     #                335     #AAG CAG TTG CTG AAC GCC    1056TT AAT ATT     #Lys Gln Leu Leu Asn Ala Gln Phe Asn Ile     #            350     #CAG GTT TTA CAG GAA CAC    1104TG ACC TGT     #Gln Val Leu Gln Glu His Leu Leu Thr Cys     #        365     #GAA GTT TGT AGA TCA TTT    1152TG CCC CGA     #Glu Val Cys Arg Ser Phe Ser Met Pro Arg     #    380     #CCT CCA GAC TTG GAA TTA    1200TT GGT TCT     #Pro Pro Asp Leu Glu Leu Val Leu Gly Ser     #400     #GTA CAC CCT CCT ACT AAT    1248TG TTA GCT     #Val His Pro Pro Thr Asn Phe Leu Leu Ala     #                415     #TAC TTC TCT TTG CAC ATA    1296CA AAC TTC     #Tyr Phe Ser Leu His Ile Pro Thr Asn Phe     #            430     #TCA CTC GCG TAC CTG AGG    1344AA GTG CAG     #Ser Leu Ala Tyr Leu Arg Glu Lys Val Gln     #        445     #AGC CCT GGA TTC CTG GTA    1392CC TTT CCC     #Ser Pro Gly Phe Leu Val Gln Ala Phe Pro     #    460     #CCT GAA GGA ACC AGT TCT    1440CA GCT CCT     #Pro Glu Gly Thr Ser Ser Thr Ala Ala Pro     #480     #CAG ATG GTT CGA TCT AGA    1488TG GCT GCT     #Gln Met Val Arg Ser Arg Arg Met Ala Ala     #                495     #CTA ATA CGA GCA CAA AAA    1536AT TTA CCA     #Leu Ile Arg Ala Gln Lys Thr Tyr Leu Pro     #            510     #AAG TTA CAA AAT ATT GCA    1584GT GTT GAC     #Lys Leu Gln Asn Ile Ala Phe Ser Val Asp     #        525     #GGA AGA CCC TAC GCA CTG    1632AT CTT TTA     #Gly Arg Pro Tyr Ala Leu Gln Asn Leu Leu     #    540     #AGC TGT GAG TCT GCA AAG    1680TC ATC AGC     #Ser Cys Glu Ser Ala Lys Ala Phe Ile Ser     #560     #GCC CAC GCC TCT GCC AAT    1728TT CTT GGA     #Ala His Ala Ser Ala Asn Ala Leu Leu Gly     #                575     #AGG GCC AGA GTA GAT CAC    1776GG TTC CCC     #Arg Ala Arg Val Asp His Pro Arg Phe Pro     #            590     #GAT GAC AGT CCT GGG GAT    1824GA TCA GAT     #Asp Asp Ser Pro Gly Asp Pro Arg Ser Asp     #        605     #AAG GGA CTG GCC TCA TTC    1872AC AAC CTC     #Lys Gly Leu Ala Ser Phe Pro Asp Asn Leu     #    620     #GGG CTG GCG TTT CCC TCT    1920CA AGC CTT     #Gly Leu Ala Phe Pro Ser Val Ala Ser Leu     #640     #CCA AGT CTT GTT GAT AGG    1968GC AGG TGG     #Pro Ser Leu Val Asp Arg Ala Ser Arg Trp     #                655     #TCT CCT GCT TAT GAG GCA    2016TT ACC TTT     #Ser Pro Ala Tyr Glu Ala Asn Phe Thr Phe     #            670     #GTC ATT GAA GAC TGT CTG    2064CC CAC AGT     #Val Ile Glu Asp Cys Leu Ser Thr His Ser     #        685     #TTA AGT GGG GTT CTT CTT    2112AT GAA CTC     #Leu Ser Gly Val Leu Leu Leu Tyr Glu Leu     #    700     #ATG GAC AGG ATT ATT CAA    2160AA GAT GTG     #Met Asp Arg Ile Ile Gln Leu Glu Asp Val     #720     #CCA TCA CCT GCT ATC CAG    2208TT AAC CAC     #Pro Ser Pro Ala Ile Gln Leu Val Asn His     #                735     #ATT AAT AGA GCA TCA AAG    2256AT GCA TAC     #Ile Asn Arg Ala Ser Lys Leu His Ala Tyr     #            750     #GGC TTT TCC TTA TTA GCC    2304AG AAC CGT     #Gly Phe Ser Leu Leu Ala Leu Lys Asn Arg     #        765     #GAG TTG TTG GAG TGC TTT    2352GA ACT CAG     #Glu Leu Leu Glu Cys Phe Arg Gly Thr Gln     #    780     #CTG GAT GAA GAA TTT GAT    2400CG ATT GGC     #Leu Asp Glu Glu Phe Asp Arg Pro Ile Gly     #800     #CAG AAG TGG TCT GTC ATT    2448AA CTG TTC     #Gln Lys Trp Ser Val Ile Met Glu Leu Phe     #                815     #TAT GAC AAT GTC CTC TTG    2496CC TCT CTC     #Tyr Asp Asn Val Leu Leu Glu Thr Ser Leu     #            830     #TTA AAC TCT TGT TCC AAG    2544TG CAA GTT     #Leu Asn Ser Cys Ser Lys Leu Leu Gln Val     #        845     #CTC TAT GTA TTA TGT AAT    2592AT GGT CTA     #Leu Tyr Val Leu Cys Asn Asp Asn Gly Leu     #    860     #AAC ATT CCT GTG AAC GAA    2640TA GAA AAG     #Asn Ile Pro Val Asn Glu Gly Leu Glu Lys     #880     #CTT TTC ATA GCA GTT ACA    2688TA CAG CAG     #Leu Phe Ile Ala Val Thr Asp Ile Gln Gln     #                895     #TAT TTT AGA GTG ATT GAA    2736GC ACA CAG     #Tyr Phe Arg Val Ile Glu Ser Gly Thr Gln     #            910     #AAT AGC AAA AAC AAG AGG    2784AT CTT CAT     #Asn Ser Lys Asn Lys Arg Gly Tyr Leu His     #        925     #AGA GTT CTC CAG GCT GCT    2832TG CAG CTT     #Arg Val Leu Gln Ala Ala Ala Leu Gln Leu     #    940     #GAC TCT GAA AGT CCA GTG    2880CC AAT CAT     #Asp Ser Glu Ser Pro Val Thr Ala Asn His     #960     #CCT CCG AAG CGG AGA AGC    2928AT TCA GTG     #Pro Pro Lys Arg Arg Ser Arg His Ser Val     #                975     #CAG ACA GAG TCT CTG CTG    2976CT CTG GCT     #Gln Thr Glu Ser Leu Leu Phe Pro Leu Ala     #            990     #CTA CAC TCT ATG ATG CAG    3024GC GAT GAG     #Leu His Ser Met Met Gln Ala Ser Asp Glu     #      10050     #GCC TCG GAG GCA GAG CTC    3072CC AGC CAG     #Ala Ser Glu Ala Glu Leu His Pro Ser Gln     #  10205     #GCT GTG AAC AGG ATT ATT    3120CC ATC TTA     #Ala Val Asn Arg Ile Ile Leu Thr Ile Leu     #               10401030 - #                1035     #ATT TTG AGA ACT CCA GAA    3168TT ATT GAC     #Ile Leu Arg Thr Pro Glu Asp Ile Ile Asp     #              10550     #CAG ACT GAA ATT TCT GAA    3216CA GTT TCT     #Gln Thr Glu Ile Ser Glu Thr Ser Val Ser     #          10705     #TAT AAT CCA TTT CAA AAA    3264CT TCT GTA     #Tyr Asn Pro Phe Gln Lys Gln Pro Ser Val     #      10850     #AAA GTG TGT ATT GGT TCA    3312AT GGC TTC     #Lys Val Cys Ile Gly Ser Leu Asp Gly Phe     #  11005     #ACT AAA ATC CTG GGG TCT    3360AG CAG TGG     #Thr Lys Ile Leu Gly Ser Lys Gln Gln Trp     #               11201110 - #                1115     #AGA TTG CTA GCG CAT ATT    3408AG CTT GGA     #Arg Leu Leu Ala His Ile Asp Gln Leu Gly     #              11350     #AAG CAG ATA CTT GAG ATA    3456AA GAA CGG     #Lys Gln Ile Leu Glu Ile Val Gln Glu Arg     #          11505     #CGT GAC TGT CTT AGC CCC    3504AT ATA CTT     #Arg Asp Cys Leu Ser Pro Gln Asp Ile Leu     #      11650     #TAT TTG TCA GAG TTG ATA    3552TG GTT TTG     #Tyr Leu Ser Glu Leu Ile Lys Leu Val Leu     #  11805     #GAA ATG GAC ACA GCA GAA    3600GT GAA GAA     #Glu Met Asp Thr Ala Glu Leu Ser Glu Glu     #               12001190 - #                1195     #CAC AAG TGC ATC CCG CCC    3648TA TGT GGC     #His Lys Cys Ile Pro Pro Lys Leu Cys Gly     #              12150     #ATC ATC AGA GAG GAG CAA    3696TC ATT AAG     #Ile Ile Arg Glu Glu Gln Glu Leu Ile Lys     #          12305     #AAA GGC TCA TGG CAG AAA    3744GT GTG AGC     #Lys Gly Ser Trp Gln Lys Glu Ser Val Ser     #      12450     #CAG AGG CTC GAT TTC AAA    3792GT CTC TTC     #Gln Arg Leu Asp Phe Lys Gln Ser Leu Phe     #  12605     #ATC ACC CAG GCT GTA TCA    3840CT GCA GAC     #Ile Thr Gln Ala Val Ser Ile Ala Ala Asp     #               12801270 - #                1275     #ATC CAG CAC ATC AGA GGG    3888AG AAG GTG     #Ile Gln His Ile Arg Gly Arg Lys Lys Val     #              12950     #CAC TGG CAG GAA TGC ATC    3936CC AGC AGG     #His Trp Gln Glu Cys Ile Ser Ala Ser Arg     #          13105     #TAT GAC CCA ATC TAC TAT    3984CA GTC TGG     #Tyr Asp Pro Ile Tyr Tyr Arg Ala Val Trp     #      13250     #GGG CCA AAC CGA GAG AGG    4032CA ACA GAA     #Gly Pro Asn Arg Glu Arg Asp Pro Thr Glu     #  13405     #CCC AAT AAG TAC CTC CTG    4080TA ACT ATT     #Pro Asn Lys Tyr Leu Leu Tyr Leu Thr Ile     #               13601350 - #                1355     #AGG CCC CCA CTC TCT TAC    4128GT GTG CTC     #Arg Pro Pro Leu Ser Tyr Glu Gly Val Leu     #              13750     #TCC TCT ACT GTC AAA GAC    4176CT TCC TTC     #Ser Ser Thr Val Lys Asp His Ser Ser Phe     #          13905     #CGA AGA TGT ATC AGT GTT    4224GA GTG AAT     #Arg Arg Cys Ile Ser Val Ile Arg Val Asn     #      14050     #TTG TTA GGT AAA TGT GGG    4272GG GAA TTG     #Leu Leu Gly Lys Cys Gly Ala Gly Glu Leu     #  14205     #GCA GTT GAA AGC TCG AGC    4320CC TCT GAC     #Ala Val Glu Ser Ser Ser Asn Ala Ser Asp     #               14401430 - #                1435     #TCT TGG ACA TAT GAG GAA    4368CA TCA TTT     #Ser Trp Thr Tyr Glu Glu Pro Ala Ser Phe     #              14550     #CTA AGA GAT AAT GCT GTA    4416GG TGG CAA     #Leu Arg Asp Asn Ala Val Arg Trp Trp Gln     #          14705     #CTA TTA GCA TTT GAC AAT    4464GA ACA CTC     #Leu Leu Ala Phe Asp Asn Gly Arg Thr Leu     #      14850     #ATC CTC ACA AAT AAC CTC    4512AC CAG AGC     #Ile Leu Thr Asn Asn Leu Val Tyr Gln Ser     #  15005     #GCT CTG ACA AAC CTG TGG    4560AC ATC ACC     #Ala Leu Thr Asn Leu Trp Gly Asn Ile Thr     #               15201510 - #                1515     #TTG ACT CAT TTA AAC AAG    4608TT GAA TAT     #Leu Thr His Leu Asn Lys Asn Phe Glu Tyr     #              15350     #CAG TAC CCG GTG TTC CCC    4656AT CTC ATG     #Gln Tyr Pro Val Phe Pro Asn Asp Leu Met     #          15505     #CTT GAC CTC AAT GAT CCA    4704GT GAG ACT     #Leu Asp Leu Asn Asp Pro Val Ser Glu Thr     #      15650     #GCT GTG CAG TAT AAA GAA    4752AG CCT ATA     #Ala Val Gln Tyr Lys Glu Ser Lys Pro Ile     #  15805     #TAC TTG GAG GAG GAG TAT    4800CA TAC AAG     #Tyr Leu Glu Glu Glu Tyr Asp Thr Tyr Lys     #               16001590 - #                1595     #CCT CCT GTG CAA CCC TAC    4848AC CCC ATG     #Pro Pro Val Gln Pro Tyr Asp Asp Pro Met     #              16150     #ACC GTG CTC CAC TTC CTG    4896AC AGC GGC     #Thr Val Leu His Phe Leu Ser Asn Ser Gly     #          16305     #CTA GCC TAT CAA GAT CAG    4944AA ATG TTT     #Leu Ala Tyr Gln Asp Gln Thr Lys Met Phe     #      16450     #TCT ACA AAC ACA ACT TGG    4992CA TTT CAT     #Ser Thr Asn Thr Thr Trp Arg Thr Phe His     #  16605     #GTG AAG GAG CTG ATT CCA    5040TG ACT GAT     #Val Lys Glu Leu Ile Pro Ser Met Thr Asp     #               16801670 - #                1675     #AAC CGT GAA GGC TTT GAC    5088TC TTA GTG     #Asn Arg Glu Gly Phe Asp Glu Phe Leu Val     #              16950     #AAC CAC GTC AAT CTT CCT    5136AG CGG GTT     #Asn His Val Asn Leu Pro Gly Glu Arg Val     #          17105     #ATC CTT ATT CAC CGG CAA    5184GG CTG TTC     #Ile Leu Ile His Arg Gln Pro Arg Leu Phe     #      17250     #ATC TGT CAC TGG ATC GAC    5232CC CAG AAC     #Ile Cys His Trp Ile Asp Val Ser Gln Asn     #  17405     #GCG TCT GTT CAA GCC ATC    5280AG GGG AAG     #Ala Ser Val Gln Ala Ile Gln Lys Gly Lys     #               17601750 - #                1755     #ATG GAT GTC TCT GCA GTT    5328AT TTT GGA     #Met Asp Val Ser Ala Val Thr Tyr Phe Gly     #              17750     #ACC ATG ATA AAA ACC TAC    5376CT TTA GAA     #Thr Met Ile Lys Thr Tyr Arg Ala Leu Glu     #          17905     #GCC CAT GCC AGC CGA CCT    5424TC CAC ACA     #Ala His Ala Ser Arg Pro Leu Phe His Thr     #      18050     #CCA GCA GCT GTT GGC TTG    5472GA GAG CTT     #Pro Ala Ala Val Gly Leu Glu Gly Glu Leu     #  18205     #GAA CCA GTC AAG GAA GTC    5520AG ACC CGA     #Glu Pro Val Lys Glu Val Arg Glu Thr Arg     #               18401830 - #                1835     #GGC TTG AAG TGG GGG GAG    5568GG ATA AAA     #Gly Leu Lys Trp Gly Glu Ser Trp Ile Lys     #              18550     #GTG GTC TGC TTC AGC CAG    5616CA GTA CCT     #Val Val Cys Phe Ser Gln Ala Pro Val Pro     #          18705     #GCA CTG CCC ACC AGA GCC    5664CC CTG CAG     #Ala Leu Pro Thr Arg Ala Gly Ser Leu Gln     #      18850     #CTG ATG ACC TAC AAC AAG    5712TC TGT CTT     #Leu Met Thr Tyr Asn Lys Asn Phe Cys Leu     #  19005     #AAT ATT CAG TGG TCT GCT    5760AC AAC ACC     #Asn Ile Gln Trp Ser Ala Met Asn Asn Thr     #               19201910 - #                1915     #TTA CGG TTG AAA AGT AAG    5808AC AAC ATC     #Leu Arg Leu Lys Ser Lys Ala Asp Asn Ile     #              19350     #AGT TCA CAG CAG CAC CAG    5856TC ATT CAG     #Ser Ser Gln Gln His Gln Asn Phe Ile Gln     #          19505     #TGT CAG CTC TTC ACT GGG    5904CT GAC AGT     #Cys Gln Leu Phe Thr Gly Val Pro Asp Ser     #      19650     #AAC AGG CTC ACC AGC AGC    5952CC TAT ACC     #Asn Arg Leu Thr Ser Ser Thr Ala Tyr Thr     #  19805     #ATG CAT CTC TAT GGA CAC    6000AG AGT CAG     #Met His Leu Tyr Gly His Met Glu Ser Gln     #               20001990 - #                1995     #AAG CCG TAC AGC GTG ATG    6048GT GTC TGC     #Lys Pro Tyr Ser Val Met Leu Cys Val Cys     #              20150     #GTA TGG GAC CTG AAC AGG    6096CC TGC ATA     #Val Trp Asp Leu Asn Arg Gly Thr Cys Ile     #          20305     #AAA AGC CCT GTG ACG GCT    6144CT GGA CAC     #Lys Ser Pro Val Thr Ala Leu Ala Gly His     #      20450     #GCT ACT GTG TGT GAC TCA    6192GT GAC ATT     #Ala Thr Val Cys Asp Ser Ser Gly Asp Ile     #  20605     #ACC GTG AAT GGG GAC CTC    6240GA CTC TGG     #Thr Val Asn Gly Asp Leu Leu Arg Leu Trp     #               20802070 - #                2075     #TGT TCT GTA GCT TTC TCC    6288AG ATC ATT     #Cys Ser Val Ala Phe Ser Arg Glu Ile Ile     #              20950     #ATT GCT GGG GGA TTA GAA    6336TC AAC GTC     #Ile Ala Gly Gly Leu Glu Ser Ile Asn Val     #          21105     #GAC TTG AAG CCT GTG AGA    6384GC ACA TGG     #Asp Leu Lys Pro Val Arg Trp Ser Thr Trp     #      21250     #ATC ATA AGC CTG ACA TTC    6432AT AAG CCC     #Ile Ile Ser Leu Thr Phe Ser Asn Lys Pro     #  21405     #AAC AGT GAG GGG ACA GTG    6480AC ACT GCC     #Asn Ser Glu Gly Thr Val Leu Tyr Thr Ala     #               21602150 - #                2155     #GTG AAG CTG CCC ATG TTC    6528AG CAG CGT     #Val Lys Leu Pro Met Phe Asp Gln Gln Arg     #              21750     #TGAAGAGAAG GAGTGTCCCC ACA  6581CA GCT GGA      Tyr Ser Phe Leu Ser Ser Tyr Ala Ala Gly     #           2185     #TGACTGAGCA  6641GCTCTGC GAGCCTGGCT CCACCAACTG CAGAAGCAGA     #CCGTGGGCCT  6701GACAACA CACGTGCCTC TGTGCGCGCT TCCCCAGCCT     #CACACACTAG  6761TGCCCTC ATTCCATAAT GGCGTGGAAG GCTGGGTCTG     #CAAAGATCAG  6821GAATCTT GATGCTTTTT CCCAAAAGGT TAGGCTGAAT     #       6830     - (2) INFORMATION FOR SEQ ID NO:2:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 2186 amino               (B) TYPE: amino acid               (C) TOPOLOGY: unknown     -     (ii) MOLECULE TYPE: protein     -      (v) FRAGMENT TYPE: internal     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:     #Gly Gln Arg Lys Thr Asper Ile Trp Val Ser     #                 15     #Thr Ser Leu Ser Ser Aspal Leu Pro Arg Lys     #             30     #Cys Leu Thr Ser Gln Gluys Met Ile Gly His     #         45     #Leu Gly Asn Leu Leu Leula Gly Lys Trp Asp     #     60     #Ala Phe Phe Leu Tyr Alale Gly Ser Gln Glu     # 80     #Cys Lys Tyr Gly Gln Prohr Ser Ile Met Pro     #                 95     #Asp Ile Leu Arg Cys Aspys Tyr Ile Asn Lys     #            110     #Glu Val Asp Val Gly Leuhe Met Thr Lys Lys     #        125     #Thr Cys Cys Pro Ala Glner Val Val Tyr Thr     #    140     #Lys Gly Gln Val Lys Thrro Val Ile Arg Leu     #160     #Glu Ala Gln Ser Ile Leuro Phe Ser Ser Lys     #                175     #Pro Thr Glu Cys Lys Alaeu Lys Gly Leu Gln     #            190     #Ala Gly Thr Phe Val Pheis Glu Ile Gly Gly     #        205     #Cys Glu Glu Thr Gln Alaal Glu Leu Ser Ser     #    220     #Lys Tyr Ser Gln Gln Argle Leu Ser Leu Ile     #240     #Ser Met Ile His Gln Valsn Cys Asn Gly Leu     #                255     #His Ile Leu Lys Thr Leuys Ile Val Gly Phe     #            270     #His Val Ser Glu His Glyly Glu Glu Val Ile     #        285     #Ile Ile Gln Asp Val Lysal Glu Ser His Ala     #    300     #Ile Trp Asn Lys Ala Glueu Leu Asp Trp Lys     #320     #Leu Glu Val Leu Ile Arghr Leu Leu Ala Ala     #                335     #Lys Gln Leu Leu Asn Alaln Gln Phe Asn Ile     #            350     #Gln Val Leu Gln Glu Hishe Leu Leu Thr Cys     #        365     #Glu Val Cys Arg Ser Phehr Ser Met Pro Arg     #    380     #Pro Pro Asp Leu Glu Leulu Val Leu Gly Ser     #400     #Val His Pro Pro Thr Asnsn Phe Leu Leu Ala     #                415     #Tyr Phe Ser Leu His Ilesn Pro Thr Asn Phe     #            430     #Ser Leu Ala Tyr Leu Argln Glu Lys Val Gln     #        445     #Ser Pro Gly Phe Leu Vally Gln Ala Phe Pro     #    460     #Pro Glu Gly Thr Ser Serhe Thr Ala Ala Pro     #480     #Gln Met Val Arg Ser Argln Arg Met Ala Ala     #                495     #Leu Ile Arg Ala Gln Lysro Thr Tyr Leu Pro     #            510     #Lys Leu Gln Asn Ile Alaly Phe Ser Val Asp     #        525     #Gly Arg Pro Tyr Ala Leuys Gln Asn Leu Leu     #    540     #Ser Cys Glu Ser Ala Lyslu Ala Phe Ile Ser     #560     #Ala His Ala Ser Ala Asnlu Ala Leu Leu Gly     #                575     #Arg Ala Arg Val Asp Hiser Pro Arg Phe Pro     #            590     #Asp Asp Ser Pro Gly Asplu Pro Arg Ser Asp     #        605     #Lys Gly Leu Ala Ser Pherg Pro Asp Asn Leu     #    620     #Gly Leu Ala Phe Pro Serhr Val Ala Ser Leu     #640     #Pro Ser Leu Val Asp Argal Ala Ser Arg Trp     #                655     #Ser Pro Ala Tyr Glu Alalu Asn Phe Thr Phe     #            670     #Val Ile Glu Asp Cys Leuhr Ser Thr His Ser     #        685     #Leu Ser Gly Val Leu Leuly Leu Tyr Glu Leu     #    700     #Met Asp Arg Ile Ile Glnet Leu Glu Asp Val     #720     #Pro Ser Pro Ala Ile Glnal Leu Val Asn His     #                735     #Ile Asn Arg Ala Ser Lyseu Leu His Ala Tyr     #            750     #Gly Phe Ser Leu Leu Alahe Leu Lys Asn Arg     #        765     #Glu Leu Leu Glu Cys Pheis Arg Gly Thr Gln     #    780     #Leu Asp Glu Glu Phe Asply Arg Pro Ile Gly     #800     #Gln Lys Trp Ser Val Ileis Met Glu Leu Phe     #                815     #Tyr Asp Asn Val Leu Leule Glu Thr Ser Leu     #            830     #Leu Asn Ser Cys Ser Lyseu Leu Leu Gln Val     #        845     #Leu Tyr Val Leu Cys Asneu Asp Asn Gly Leu     #    860     #Asn Ile Pro Val Asn Glusn Gly Leu Glu Lys     #880     #Leu Phe Ile Ala Val Thrys Asp Ile Gln Gln     #                895     #Tyr Phe Arg Val Ile Gluer Ser Gly Thr Gln     #            910     #Asn Ser Lys Asn Lys Argeu Gly Tyr Leu His     #        925     #Arg Val Leu Gln Ala Alaeu Ala Leu Gln Leu     #    940     #Asp Ser Glu Ser Pro Valer Thr Ala Asn His     #960     #Pro Pro Lys Arg Arg Seris Arg His Ser Val     #                975     #Gln Thr Glu Ser Leu Leuys Phe Pro Leu Ala     #            990     #Leu His Ser Met Met Glnal Ala Ser Asp Glu     #      10050     #Ala Ser Glu Ala Glu Leulu His Pro Ser Gln     #  10205     #Ala Val Asn Arg Ile Ilerg Leu Thr Ile Leu      025                1 - #030                1035 - #                1040     #Ile Leu Arg Thr Pro Gluer Asp Ile Ile Asp     #              10550     #Gln Thr Glu Ile Ser Gluys Thr Ser Val Ser     #          10705     #Tyr Asn Pro Phe Gln Lyslu Gln Pro Ser Val     #      10850     #Lys Val Cys Ile Gly Sereu Leu Asp Gly Phe     #  11005     #Thr Lys Ile Leu Gly Serer Lys Gln Gln Trp     #               11201110 - #                1115     #Arg Leu Leu Ala His Ilerg Asp Gln Leu Gly     #              11350     #Lys Gln Ile Leu Glu Ilehr Val Gln Glu Arg     #          11505     #Arg Asp Cys Leu Ser Prois Gln Asp Ile Leu     #      11650     #Tyr Leu Ser Glu Leu Ilela Lys Leu Val Leu     #  11805     #Glu Met Asp Thr Ala Glulu Leu Ser Glu Glu     #               12001190 - #                1195     #His Lys Cys Ile Pro Proeu Lys Leu Cys Gly     #              12150     #Ile Ile Arg Glu Glu Glnro Glu Leu Ile Lys     #          12305     #Lys Gly Ser Trp Gln Lyslu Glu Ser Val Ser     #      12450     #Gln Arg Leu Asp Phe Lysln Gln Ser Leu Phe     #  12605     #Ile Thr Gln Ala Val Serys Ile Ala Ala Asp     #               12801270 - #                1275     #Ile Gln His Ile Arg Glylu Arg Lys Lys Val     #              12950     #His Trp Gln Glu Cys Ileeu Ser Ala Ser Arg     #          13105     #Tyr Asp Pro Ile Tyr Tyrsp Arg Ala Val Trp     #      13250     #Gly Pro Asn Arg Glu Argeu Asp Pro Thr Glu     #  13405     #Pro Asn Lys Tyr Leu Leuys Tyr Leu Thr Ile     #               13601350 - #                1355     #Arg Pro Pro Leu Ser Tyrer Glu Gly Val Leu     #              13750     #Ser Ser Thr Val Lys Asphr His Ser Ser Phe     #          13905     #Arg Arg Cys Ile Ser Valer Ile Arg Val Asn     #      14050     #Leu Leu Gly Lys Cys Glyhr Ala Gly Glu Leu     #  14205     #Ala Val Glu Ser Ser Sersp Asn Ala Ser Asp     #               14401430 - #                1435     #Ser Trp Thr Tyr Glu Glulu Pro Ala Ser Phe     #              14550     #Leu Arg Asp Asn Ala Valrg Arg Trp Trp Gln     #          14705     #Leu Leu Ala Phe Asp Asnsn Gly Arg Thr Leu     #      14850     #Ile Leu Thr Asn Asn Leusp Val Tyr Gln Ser     #  15005     #Ala Leu Thr Asn Leu Trpyr Gly Asn Ile Thr     #               15201510 - #                1515     #Leu Thr His Leu Asn Lyshr Asn Phe Glu Tyr     #              15350     #Gln Tyr Pro Val Phe Prohe Asn Asp Leu Met     #          15505     #Leu Asp Leu Asn Asp Proyr Val Ser Glu Thr     #      15650     #Ala Val Gln Tyr Lys Glueu Ser Lys Pro Ile     #  15805     #Tyr Leu Glu Glu Glu Tyral Asp Thr Tyr Lys     #               16001590 - #                1595     #Pro Pro Val Gln Pro Tyrlu Asp Asp Pro Met     #              16150     #Thr Val Leu His Phe Leuyr Ser Asn Ser Gly     #          16305     #Leu Ala Tyr Gln Asp Glnhe Thr Lys Met Phe     #      16450     #Ser Thr Asn Thr Thr Trpsp Arg Thr Phe His     #  16605     #Val Lys Glu Leu Ile Prolu Ser Met Thr Asp     #               16801670 - #                1675     #Asn Arg Glu Gly Phe Aspro Glu Phe Leu Val     #              16950     #Asn His Val Asn Leu Prosn Gly Glu Arg Val     #          17105     #Ile Leu Ile His Arg Glnsp Pro Arg Leu Phe     #      17250     #Ile Cys His Trp Ile Aspis Val Ser Gln Asn     #  17405     #Ala Ser Val Gln Ala Ileys Gln Lys Gly Lys     #               17601750 - #                1755     #Met Asp Val Ser Ala Valla Thr Tyr Phe Gly     #              17750     #Thr Met Ile Lys Thr Tyrrg Arg Ala Leu Glu     #          17905     #Ala His Ala Ser Arg Proln Leu Phe His Thr     #      18050     #Pro Ala Ala Val Gly Leule Glu Gly Glu Leu     #  18205     #Glu Pro Val Lys Glu Valhe Arg Glu Thr Arg     #               18401830 - #                1835     #Gly Leu Lys Trp Gly Glueu Ser Trp Ile Lys     #              18550     #Val Val Cys Phe Ser Glner Ala Pro Val Pro     #          18705     #Ala Leu Pro Thr Arg Alahe Gly Ser Leu Gln     #      18850     #Leu Met Thr Tyr Asn Lysrg Asn Phe Cys Leu     #  19005     #Asn Ile Gln Trp Ser Alaer Met Asn Asn Thr     #               19201910 - #                1915     #Leu Arg Leu Lys Ser Lysyr Ala Asp Asn Ile     #              19350     #Ser Ser Gln Gln His Glnle Asn Phe Ile Gln     #          19505     #Cys Gln Leu Phe Thr Glyrp Val Pro Asp Ser     #      19650     #Asn Arg Leu Thr Ser Serle Thr Ala Tyr Thr     #  19805     #Met His Leu Tyr Gly Hislu Met Glu Ser Gln     #               20001990 - #                1995     #Lys Pro Tyr Ser Val Metly Leu Cys Val Cys     #              20150     #Val Trp Asp Leu Asn Argsp Gly Thr Cys Ile     #          20305     #Lys Ser Pro Val Thr Alaer Leu Ala Gly His     #      20450     #Ala Thr Val Cys Asp Serhr Ser Gly Asp Ile     #  20605     #Thr Val Asn Gly Asp Leusp Leu Arg Leu Trp     #               20802070 - #                2075     #Cys Ser Val Ala Phe Serys Arg Glu Ile Ile     #              20950     #Ile Ala Gly Gly Leu Glual Ser Ile Asn Val     #          21105     #Asp Leu Lys Pro Val Argeu Trp Ser Thr Trp     #      21250     #Ile Ile Ser Leu Thr Pheys Ser Asn Lys Pro     #  21405     #Asn Ser Glu Gly Thr Valis Leu Tyr Thr Ala     #               21602150 - #                2155     #Val Lys Leu Pro Met Pheys Asp Gln Gln Arg     #              21750     -  Tyr Ser Phe Leu Ser Ser Tyr Ala Ala Gly     #           2185     - (2) INFORMATION FOR SEQ ID NO:3:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 11 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:     #       11     - (2) INFORMATION FOR SEQ ID NO:4:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 11 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:     #       11     - (2) INFORMATION FOR SEQ ID NO:5:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 11 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:     #       11     - (2) INFORMATION FOR SEQ ID NO:6:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 11 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:     #       11     - (2) INFORMATION FOR SEQ ID NO:7:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 14 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:     #     14     - (2) INFORMATION FOR SEQ ID NO:8:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 13 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:     #      13     - (2) INFORMATION FOR SEQ ID NO:9:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 12616 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (ix) FEATURE:               (A) NAME/KEY: CDS               (B) LOCATION: 190..11592     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:     - GCGGCCGCGT CGACGCGGCG GCGGCAGCGG CGTCGGCTCG GGGTTCTCCG GG - #AGAGGGGG       60     - AGTGCGCGGC GGCCGCAGCT GCCACAAACC AGGTGAAGCT TTGTTCTAAG AA - #TATTTGTT      120     - TCATCTAGTT TATGAGTCCA AATGATATAG ACTGTAAATG TCACAGCAGT GG - #TGAAAGAC      180     - TGCTCGGTC ATG AGC ACC GAC AGT AAC TCA CTG GCA - # CGT GAA TTT CTG      228     #Asn Ser Leu Ala Arg Glu Phe Leu     #         10     - ACC GAT GTC AAC CGG CTT TGC AAT GCA GTG GT - #C CAG AGG GTG GAG GCC      276     Thr Asp Val Asn Arg Leu Cys Asn Ala Val Va - #l Gln Arg Val Glu Ala     #     25     - AGG GAG GAA GAA GAG GAG GAG ACG CAC ATG GC - #A ACC CTT GGA CAG TAC      324     Arg Glu Glu Glu Glu Glu Glu Thr His Met Al - #a Thr Leu Gly Gln Tyr     # 45     - CTT GTC CAT GGT CGA GGA TTT CTA TTA CTT AC - #C AAG CTA AAT TCT ATA      372     Leu Val His Gly Arg Gly Phe Leu Leu Leu Th - #r Lys Leu Asn Ser Ile     #                 60     - ATT GAT CAG GCA TTG ACA TGT AGA GAA GAA CT - #C CTG ACT CTT CTT CTG      420     Ile Asp Gln Ala Leu Thr Cys Arg Glu Glu Le - #u Leu Thr Leu Leu Leu     #             75     - TCT CTC CTT CCA CTG GTA TGG AAG ATA CCT GT - #C CAA GAA GAA AAG GCA      468     Ser Leu Leu Pro Leu Val Trp Lys Ile Pro Va - #l Gln Glu Glu Lys Ala     #         90     - ACA GAT TTT AAC CTA CCG CTC TCA GCA GAT AT - #A ATC CTG ACC AAA GAA      516     Thr Asp Phe Asn Leu Pro Leu Ser Ala Asp Il - #e Ile Leu Thr Lys Glu     #    105     - AAG AAC TCA AGT TCA CAA AGA TCC ACT CAG GA - #A AAA TTA CAT TTA GAA      564     Lys Asn Ser Ser Ser Gln Arg Ser Thr Gln Gl - #u Lys Leu His Leu Glu     110                 1 - #15                 1 - #20                 1 -     #25     - GGA AGT GCC CTG TCT AGT CAG GTT TCT GCA AA - #A GTA AAT GTT TTT CGA      612     Gly Ser Ala Leu Ser Ser Gln Val Ser Ala Ly - #s Val Asn Val Phe Arg     #               140     - AAA AGC AGA CGA CAG CGT AAA ATT ACC CAT CG - #C TAT TCT GTA AGA GAT      660     Lys Ser Arg Arg Gln Arg Lys Ile Thr His Ar - #g Tyr Ser Val Arg Asp     #           155     - GCA AGA AAG ACA CAG CTC TCC ACC TCA GAT TC - #A GAA GCC AAT TCA GAT      708     Ala Arg Lys Thr Gln Leu Ser Thr Ser Asp Se - #r Glu Ala Asn Ser Asp     #       170     - GAA AAA GGC ATA GCA ATG AAT AAG CAT AGA AG - #G CCC CAT CTG CTG CAT      756     Glu Lys Gly Ile Ala Met Asn Lys His Arg Ar - #g Pro His Leu Leu His     #   185     - CAT TTT TTA ACA TCG TTT CCT AAA CAA GAC CA - #C CCC AAA GCT AAA CTT      804     His Phe Leu Thr Ser Phe Pro Lys Gln Asp Hi - #s Pro Lys Ala Lys Leu     190                 1 - #95                 2 - #00                 2 -     #05     - GAC CGC TTA GCA ACC AAA GAA CAG ACT CCT CC - #A GAT GCT ATG GCT TTG      852     Asp Arg Leu Ala Thr Lys Glu Gln Thr Pro Pr - #o Asp Ala Met Ala Leu     #               220     - GAA AAT TCC AGA GAG ATT ATT CCA AGA CAG GG - #G TCA AAC ACT GAC ATT      900     Glu Asn Ser Arg Glu Ile Ile Pro Arg Gln Gl - #y Ser Asn Thr Asp Ile     #           235     - TTA AGT GAG CCA GCT GCC TTG TCT GTT ATC AG - #T AAC ATG AAC AAT TCT      948     Leu Ser Glu Pro Ala Ala Leu Ser Val Ile Se - #r Asn Met Asn Asn Ser     #       250     - CCA TTT GAC TTA TGT CAT GTT TTG TTA TCT TT - #A TTA GAA AAA GTT TGT      996     Pro Phe Asp Leu Cys His Val Leu Leu Ser Le - #u Leu Glu Lys Val Cys     #   265     - AAG TTT GAC GTT ACC TTG AAT CAT AAT TCT CC - #T TTA GCA GCC AGT GTA     1044     Lys Phe Asp Val Thr Leu Asn His Asn Ser Pr - #o Leu Ala Ala Ser Val     270                 2 - #75                 2 - #80                 2 -     #85     - GTG CCC ACA CTA ACT GAA TTC CTA GCA GGC TT - #T GGG GAC TGC TGC AGT     1092     Val Pro Thr Leu Thr Glu Phe Leu Ala Gly Ph - #e Gly Asp Cys Cys Ser     #               300     - CTG AGC GAC AAC TTG GAG AGT CGA GTA GTT TC - #T GCA GGT TGG ACC GAA     1140     Leu Ser Asp Asn Leu Glu Ser Arg Val Val Se - #r Ala Gly Trp Thr Glu     #           315     - GAA CCG GTG GCT TTG ATT CAA AGG ATG CTC TT - #T CGA ACA GTG TTG CAT     1188     Glu Pro Val Ala Leu Ile Gln Arg Met Leu Ph - #e Arg Thr Val Leu His     #       330     - CTT CTG TCA GTA GAT GTT AGT ACT GCA GAG AT - #G ATG CCA GAA AAT CTT     1236     Leu Leu Ser Val Asp Val Ser Thr Ala Glu Me - #t Met Pro Glu Asn Leu     #   345     - AGG AAA AAT TTA ACT GAA TTG CTT AGA GCA GC - #T TTA AAA ATT AGA ATA     1284     Arg Lys Asn Leu Thr Glu Leu Leu Arg Ala Al - #a Leu Lys Ile Arg Ile     350                 3 - #55                 3 - #60                 3 -     #65     - TGC CTA GAA AAG CAG CCT GAC CCT TTT GCA CC - #A AGA CAA AAG AAA ACA     1332     Cys Leu Glu Lys Gln Pro Asp Pro Phe Ala Pr - #o Arg Gln Lys Lys Thr     #               380     - CTG CAG GAG GTT CAG GAA GAT TTT GTG TTT TC - #A AAG TAT CGT CAT AGA     1380     Leu Gln Glu Val Gln Glu Asp Phe Val Phe Se - #r Lys Tyr Arg His Arg     #           395     - GCC CTT CTT TTA CCT GAG CTT TTG GAA GGA GT - #T CTT CAG ATT CTG ATC     1428     Ala Leu Leu Leu Pro Glu Leu Leu Glu Gly Va - #l Leu Gln Ile Leu Ile     #       410     - TGT TGT CTT CAA AGT GCA GCT TCA AAT CCC TT - #C TAC TTC AGT CAA GCC     1476     Cys Cys Leu Gln Ser Ala Ala Ser Asn Pro Ph - #e Tyr Phe Ser Gln Ala     #   425     - ATG GAT TTG GTT CAA GAA TTC ATT CAG CAT CA - #T GGA TTT AAT TTA TTT     1524     Met Asp Leu Val Gln Glu Phe Ile Gln His Hi - #s Gly Phe Asn Leu Phe     430                 4 - #35                 4 - #40                 4 -     #45     - GAA ACA GCA GTT CTT CAA ATG GAA TGG CTG GT - #T TTA AGA GAT GGA GTT     1572     Glu Thr Ala Val Leu Gln Met Glu Trp Leu Va - #l Leu Arg Asp Gly Val     #               460     - CCT CCC GAG GCC TCA GAG CAT TTG AAA GCC CT - #A ATA AAT AGT GTG ATG     1620     Pro Pro Glu Ala Ser Glu His Leu Lys Ala Le - #u Ile Asn Ser Val Met     #           475     - AAA ATA ATG AGC ACT GTC AAA AAA GTG AAA TC - #A GAG CAA CTT CAT CAT     1668     Lys Ile Met Ser Thr Val Lys Lys Val Lys Se - #r Glu Gln Leu His His     #       490     - TCG ATG TGT ACA AGA AAA AGG CAC AGA CGA TG - #T GAA TAT TCT CAT TTT     1716     Ser Met Cys Thr Arg Lys Arg His Arg Arg Cy - #s Glu Tyr Ser His Phe     #   505     - ATG CAT CAT CAC CGA GAT CTC TCA GGT CTT CT - #G GTT TCG GCT TTT AAA     1764     Met His His His Arg Asp Leu Ser Gly Leu Le - #u Val Ser Ala Phe Lys     510                 5 - #15                 5 - #20                 5 -     #25     - AAC CAG GTT TCC AAA AAC CCA TTT GAA GAG AC - #T GCA GAT GGA GAT GTT     1812     Asn Gln Val Ser Lys Asn Pro Phe Glu Glu Th - #r Ala Asp Gly Asp Val     #               540     - TAT TAT CCT GAG CGG TGC TGT TGC ATT GCA GT - #G TGT GCC CAT CAG TGC     1860     Tyr Tyr Pro Glu Arg Cys Cys Cys Ile Ala Va - #l Cys Ala His Gln Cys     #           555     - TTG CGC TTA CTA CAG CAG GCT TCC TTG AGC AG - #C ACT TGT GTC CAG ATC     1908     Leu Arg Leu Leu Gln Gln Ala Ser Leu Ser Se - #r Thr Cys Val Gln Ile     #       570     - CTA TCG GGT GTT CAT AAC ATT GGA ATA TGC TG - #T TGT ATG GAT CCC AAA     1956     Leu Ser Gly Val His Asn Ile Gly Ile Cys Cy - #s Cys Met Asp Pro Lys     #   585     - TCT GTA ATC ATT CCT TTG CTC CAT GCT TTT AA - #A TTG CCA GCA CTG AAA     2004     Ser Val Ile Ile Pro Leu Leu His Ala Phe Ly - #s Leu Pro Ala Leu Lys     590                 5 - #95                 6 - #00                 6 -     #05     - AAT TTT CAG CAG CAT ATA TTG AAT ATC CTT AA - #C AAA CTT ATT TTG GAT     2052     Asn Phe Gln Gln His Ile Leu Asn Ile Leu As - #n Lys Leu Ile Leu Asp     #               620     - CAG TTA GGA GGA GCA GAG ATA TCA CCA AAA AT - #T AAA AAA GCA GCT TGT     2100     Gln Leu Gly Gly Ala Glu Ile Ser Pro Lys Il - #e Lys Lys Ala Ala Cys     #           635     - AAT ATT TGT ACT GTT GAC TCT GAC CAA CTA GC - #C CAA TTA GAA GAG ACA     2148     Asn Ile Cys Thr Val Asp Ser Asp Gln Leu Al - #a Gln Leu Glu Glu Thr     #       650     - CTG CAG GGA AAC TTA TGT GAT GCT GAA CTC TC - #C TCA AGT TTA TCC AGT     2196     Leu Gln Gly Asn Leu Cys Asp Ala Glu Leu Se - #r Ser Ser Leu Ser Ser     #   665     - CCT TCT TAC AGA TTT CAA GGG ATC CTG CCC AG - #C AGT GGA TCT GAA GAT     2244     Pro Ser Tyr Arg Phe Gln Gly Ile Leu Pro Se - #r Ser Gly Ser Glu Asp     670                 6 - #75                 6 - #80                 6 -     #85     - TTG TTG TGG AAA TGG GAT GCT TTA AAG GCT TA - #T CAG AAC TTT GTT TTT     2292     Leu Leu Trp Lys Trp Asp Ala Leu Lys Ala Ty - #r Gln Asn Phe Val Phe     #               700     - GAA GAA GAC AGA TTA CAT AGT ATA CAG ATT GC - #A AAT CAC ATT TGC AAT     2340     Glu Glu Asp Arg Leu His Ser Ile Gln Ile Al - #a Asn His Ile Cys Asn     #           715     - TTA ATC CAG AAA GGC AAT ATA GTT GTT CAG TG - #G AAA TTA TAT AAT TAC     2388     Leu Ile Gln Lys Gly Asn Ile Val Val Gln Tr - #p Lys Leu Tyr Asn Tyr     #       730     - ATA TTT AAT CCT GTG CTC CAA AGA GGA GTT GA - #A TTA GCA CAT CAT TGT     2436     Ile Phe Asn Pro Val Leu Gln Arg Gly Val Gl - #u Leu Ala His His Cys     #   745     - CAA CAC CTA AGC GTT ACT TCA GCT CAA AGT CA - #T GTA TGT AGC CAT CAT     2484     Gln His Leu Ser Val Thr Ser Ala Gln Ser Hi - #s Val Cys Ser His His     750                 7 - #55                 7 - #60                 7 -     #65     - AAC CAG TGC TTG CCT CAG GAC GTG CTT CAG AT - #T TAT GTA AAA ACT CTG     2532     Asn Gln Cys Leu Pro Gln Asp Val Leu Gln Il - #e Tyr Val Lys Thr Leu     #               780     - CCT ATC CTG CTT AAA TCC AGG GTA ATA AGA GA - #T TTG TTT TTG AGT TGT     2580     Pro Ile Leu Leu Lys Ser Arg Val Ile Arg As - #p Leu Phe Leu Ser Cys     #           795     - AAT GGA GTA AGT CAA ATA ATC GAA TTA AAT TG - #C TTA AAT GGT ATT CGA     2628     Asn Gly Val Ser Gln Ile Ile Glu Leu Asn Cy - #s Leu Asn Gly Ile Arg     #       810     - AGT CAT TCT CTA AAA GCA TTT GAA ACT CTG AT - #A ATC AGC CTA GGG GAG     2676     Ser His Ser Leu Lys Ala Phe Glu Thr Leu Il - #e Ile Ser Leu Gly Glu     #   825     - CAA CAG AAA GAT GCC TCA GTT CCA GAT ATT GA - #T GGG ATA GAC ATT GAA     2724     Gln Gln Lys Asp Ala Ser Val Pro Asp Ile As - #p Gly Ile Asp Ile Glu     830                 8 - #35                 8 - #40                 8 -     #45     - CAG AAG GAG TTG TCC TCT GTA CAT GTG GGT AC - #T TCT TTT CAT CAT CAG     2772     Gln Lys Glu Leu Ser Ser Val His Val Gly Th - #r Ser Phe His His Gln     #               860     - CAA GCT TAT TCA GAT TCT CCT CAG AGT CTC AG - #C AAA TTT TAT GCT GGC     2820     Gln Ala Tyr Ser Asp Ser Pro Gln Ser Leu Se - #r Lys Phe Tyr Ala Gly     #           875     - CTC AAA GAA GCT TAT CCA AAG AGA CGG AAG AC - #T GTT AAC CAA GAT GTT     2868     Leu Lys Glu Ala Tyr Pro Lys Arg Arg Lys Th - #r Val Asn Gln Asp Val     #       890     - CAT ATC AAC ACA ATA AAC CTA TTC CTC TGT GT - #G GCT TTT TTA TGC GTA     2916     His Ile Asn Thr Ile Asn Leu Phe Leu Cys Va - #l Ala Phe Leu Cys Val     #   905     - AGT AAA GAA GCA GAG TCT GAC AGG GAG TCG GC - #C AAT GAC TCA GAA GAT     2964     Ser Lys Glu Ala Glu Ser Asp Arg Glu Ser Al - #a Asn Asp Ser Glu Asp     910                 9 - #15                 9 - #20                 9 -     #25     - ACT TCT GGC TAT GAC AGC ACA GCC AGC GAG CC - #T TTA AGT CAT ATG CTG     3012     Thr Ser Gly Tyr Asp Ser Thr Ala Ser Glu Pr - #o Leu Ser His Met Leu     #               940     - CCA TGT ATA TCT CTC GAG AGC CTT GTC TTG CC - #T TCT CCT GAA CAT ATG     3060     Pro Cys Ile Ser Leu Glu Ser Leu Val Leu Pr - #o Ser Pro Glu His Met     #           955     - CAC CAA GCA GCA GAC ATT TGG TCT ATG TGT CG - #T TGG ATC TAC ATG TTG     3108     His Gln Ala Ala Asp Ile Trp Ser Met Cys Ar - #g Trp Ile Tyr Met Leu     #       970     - AGT TCA GTG TTC CAG AAA CAG TTT TAT AGG CT - #T GGT GGT TTC CGA GTA     3156     Ser Ser Val Phe Gln Lys Gln Phe Tyr Arg Le - #u Gly Gly Phe Arg Val     #   985     - TGC CAT AAG TTA ATA TTT ATG ATA ATA CAG AA - #A CTG TTC AGA AGT CAC     3204     Cys His Lys Leu Ile Phe Met Ile Ile Gln Ly - #s Leu Phe Arg Ser His     990                 9 - #95                 1 - #000                1005     - AAA GAG GAG CAA GGA AAA AAG GAG GGA GAT AC - #A AGT GTA AAT GAA AAC     3252     Lys Glu Glu Gln Gly Lys Lys Glu Gly Asp Th - #r Ser Val Asn Glu Asn     #              10205     - CAG GAT TTA AAC AGA ATT TCT CAA CCT AAG AG - #A ACT ATG AAG GAA GAT     3300     Gln Asp Leu Asn Arg Ile Ser Gln Pro Lys Ar - #g Thr Met Lys Glu Asp     #          10350     - TTA TTA TCT TTG GCT ATA AAA AGT GAC CCC AT - #A CCA TCA GAA CTA GGT     3348     Leu Leu Ser Leu Ala Ile Lys Ser Asp Pro Il - #e Pro Ser Glu Leu Gly     #      10505     - AGT CTA AAA AAG AGT GCT GAC AGT TTA GGT AA - #A TTA GAG TTA CAG CAT     3396     Ser Leu Lys Lys Ser Ala Asp Ser Leu Gly Ly - #s Leu Glu Leu Gln His     #  10650     - ATT TCT TCC ATA AAT GTG GAA GAA GTT TCA GC - #T ACT GAA GCC GCT CCC     3444     Ile Ser Ser Ile Asn Val Glu Glu Val Ser Al - #a Thr Glu Ala Ala Pro     #               10851075 - #                1080     - GAG GAA GCA AAG CTA TTT ACA AGT CAA GAA AG - #T GAG ACC TCA CTT CAA     3492     Glu Glu Ala Lys Leu Phe Thr Ser Gln Glu Se - #r Glu Thr Ser Leu Gln     #              11005     - AGT ATA CGA CTT TTG GAA GCC CTT CTG GCC AT - #T TGT CTT CAT GGT GCC     3540     Ser Ile Arg Leu Leu Glu Ala Leu Leu Ala Il - #e Cys Leu His Gly Ala     #          11150     - AGA ACT AGT CAA CAG AAG ATG GAA TTG GAG TT - #A CCT AAT CAG AAC TTG     3588     Arg Thr Ser Gln Gln Lys Met Glu Leu Glu Le - #u Pro Asn Gln Asn Leu     #      11305     - TCT GTG GAA AGT ATA TTA TTT GAA ATG AGG GA - #C CAT CTT TCC CAG TCA     3636     Ser Val Glu Ser Ile Leu Phe Glu Met Arg As - #p His Leu Ser Gln Ser     #  11450     - AAG GTG ATT GAA ACA CAA CTA GCA AAG CCG TT - #A TTT GAT GCC CTG CTT     3684     Lys Val Ile Glu Thr Gln Leu Ala Lys Pro Le - #u Phe Asp Ala Leu Leu     #               11651155 - #                1160     - CGA GTT GCC CTC GGG AAT TAT TCA GCA GAT TT - #T GAA CAT AAT GAT GCT     3732     Arg Val Ala Leu Gly Asn Tyr Ser Ala Asp Ph - #e Glu His Asn Asp Ala     #              11805     - ATG ACT GAG AAG AGT CAT CAA TCT GCA GAA GA - #A TTG TCA TCC CAG CCT     3780     Met Thr Glu Lys Ser His Gln Ser Ala Glu Gl - #u Leu Ser Ser Gln Pro     #          11950     - GGT GAT TTT TCA GAA GAA GCT GAG GAT TCT CA - #G TGT TGT AGT TTT AAA     3828     Gly Asp Phe Ser Glu Glu Ala Glu Asp Ser Gl - #n Cys Cys Ser Phe Lys     #      12105     - CTT TTA GTT GAA GAA GAA GGT TAC GAA GCA GA - #T AGT GAA AGC AAT CCT     3876     Leu Leu Val Glu Glu Glu Gly Tyr Glu Ala As - #p Ser Glu Ser Asn Pro     #  12250     - GAA GAT GGC GAA ACC CAG GAT GAT GGG GTA GA - #C TTA AAG TCT GAA ACA     3924     Glu Asp Gly Glu Thr Gln Asp Asp Gly Val As - #p Leu Lys Ser Glu Thr     #               12451235 - #                1240     - GAA GGT TTC AGT GCA TCA AGC AGT CCA AAT GA - #C TTA CTC GAA AAC CTC     3972     Glu Gly Phe Ser Ala Ser Ser Ser Pro Asn As - #p Leu Leu Glu Asn Leu     #              12605     - ACT CAA GGG GAA ATA ATT TAT CCT GAG ATT TG - #T ATG CTG GAA TTA AAT     4020     Thr Gln Gly Glu Ile Ile Tyr Pro Glu Ile Cy - #s Met Leu Glu Leu Asn     #          12750     - TTG CTT TCT GCT AGT AAA GCC AAA CTT GAT GT - #G CTT GCC CAT GTA TTT     4068     Leu Leu Ser Ala Ser Lys Ala Lys Leu Asp Va - #l Leu Ala His Val Phe     #      12905     - GAG AGT TTT TTG AAA ATT ATT AGG CAG AAA GA - #A AAG AAT GTT TTT CTG     4116     Glu Ser Phe Leu Lys Ile Ile Arg Gln Lys Gl - #u Lys Asn Val Phe Leu     #  13050     - CTC ATG CAA CAG GGA ACT GTG AAA AAT CTT TT - #A GGA GGG TTC TTG AGT     4164     Leu Met Gln Gln Gly Thr Val Lys Asn Leu Le - #u Gly Gly Phe Leu Ser     #               13251315 - #                1320     - ATT TTA ACA CAG GAT GAT TCT GAT TTT CAA GC - #A TGC CAG AGA GTA TTG     4212     Ile Leu Thr Gln Asp Asp Ser Asp Phe Gln Al - #a Cys Gln Arg Val Leu     #              13405     - GTG GAT CTT TTG GTA TCT TTG ATG AGT TCA AG - #A ACA TGT TCA GAA GAG     4260     Val Asp Leu Leu Val Ser Leu Met Ser Ser Ar - #g Thr Cys Ser Glu Glu     #          13550     - CTA ACC CTT CTT TTG AGA ATA TTT CTG GAG AA - #A TCT CCT TGT ACA AAA     4308     Leu Thr Leu Leu Leu Arg Ile Phe Leu Glu Ly - #s Ser Pro Cys Thr Lys     #      13705     - ATT CTT CTT CTG GGT ATT CTG AAA ATT ATT GA - #A AGT GAT ACT ACT ATG     4356     Ile Leu Leu Leu Gly Ile Leu Lys Ile Ile Gl - #u Ser Asp Thr Thr Met     #  13850     - AGC CCT TCA CAG TAT CTA ACC TTC CCT TTA CT - #G CAC GCT CCA AAT TTA     4404     Ser Pro Ser Gln Tyr Leu Thr Phe Pro Leu Le - #u His Ala Pro Asn Leu     #               14051395 - #                1400     - AGC AAC GGT GTT TCA TCA CAA AAG TAT CCT GG - #G ATT TTA AAC AGT AAG     4452     Ser Asn Gly Val Ser Ser Gln Lys Tyr Pro Gl - #y Ile Leu Asn Ser Lys     #              14205     - GCC ATG GGT TTA TTG AGA AGA GCA CGA GTT TC - #A CGG AGC AAG AAA GAG     4500     Ala Met Gly Leu Leu Arg Arg Ala Arg Val Se - #r Arg Ser Lys Lys Glu     #          14350     - GCT GAT AGA GAG AGT TTT CCC CAT CGG CTG CT - #T TCA TCT TGG CAC ATA     4548     Ala Asp Arg Glu Ser Phe Pro His Arg Leu Le - #u Ser Ser Trp His Ile     #      14505     - GCC CCA GTC CAC CTG CCG TTG CTG GGG CAA AA - #C TGC TGG CCA CAC CTA     4596     Ala Pro Val His Leu Pro Leu Leu Gly Gln As - #n Cys Trp Pro His Leu     #  14650     - TCA GAA GGT TTC AGT GTT TCC CTG TGG TTT AA - #T GTG GAG TGT ATC CAT     4644     Ser Glu Gly Phe Ser Val Ser Leu Trp Phe As - #n Val Glu Cys Ile His     #               14851475 - #                1480     - GAA GCT GAG AGT ACT ACA GAA AAA GGA AAG AA - #G ATA AAG AAA AGA AAC     4692     Glu Ala Glu Ser Thr Thr Glu Lys Gly Lys Ly - #s Ile Lys Lys Arg Asn     #              15005     - AAA TCA TTA ATT TTA CCA GAT AGC AGT TTT GA - #T GGT ACA GAG AGC GAC     4740     Lys Ser Leu Ile Leu Pro Asp Ser Ser Phe As - #p Gly Thr Glu Ser Asp     #          15150     - AGA CCA GAA GGT GCA GAG TAC ATA AAT CCT GG - #T GAA AGA CTC ATA GAA     4788     Arg Pro Glu Gly Ala Glu Tyr Ile Asn Pro Gl - #y Glu Arg Leu Ile Glu     #      15305     - GAA GGA TGT ATT CAT ATA ATT TCA CTG GGA TC - #C AAA GCG TTG ATG ATC     4836     Glu Gly Cys Ile His Ile Ile Ser Leu Gly Se - #r Lys Ala Leu Met Ile     #  15450     - CAA GTG TGG GCT GAT CCC CAC AAT GCC ACT CT - #T ATC TTT CGT GTG TGC     4884     Gln Val Trp Ala Asp Pro His Asn Ala Thr Le - #u Ile Phe Arg Val Cys     #               15651555 - #                1560     - ATG GAT TCA AAT GAT GAC ATG AAA GCT GTT TT - #A CTA GCA CAG GTT GAA     4932     Met Asp Ser Asn Asp Asp Met Lys Ala Val Le - #u Leu Ala Gln Val Glu     #              15805     - TCA CAG GAG AAT ATT TTC CTC CCA AGC AAA TG - #G CAA CAT TTA GTA CTC     4980     Ser Gln Glu Asn Ile Phe Leu Pro Ser Lys Tr - #p Gln His Leu Val Leu     #          15950     - ACC TAC TTA CAG CAG CCC CAA GGG AAA AGG AG - #G ATT CAT GGG AAA ATC     5028     Thr Tyr Leu Gln Gln Pro Gln Gly Lys Arg Ar - #g Ile His Gly Lys Ile     #      16105     - TCC ATA TGG GTC TCT GGA CAG AGG AAG CCT GA - #T GTT ACT TTG GAT TTT     5076     Ser Ile Trp Val Ser Gly Gln Arg Lys Pro As - #p Val Thr Leu Asp Phe     #  16250     - ATG CTT CCA AGA AAA ACA AGT TTG TCA TCT GA - #T AGC AAT AAA ACA TTT     5124     Met Leu Pro Arg Lys Thr Ser Leu Ser Ser As - #p Ser Asn Lys Thr Phe     #               16451635 - #                1640     - TGC ATG ATT GGC CAT TGT TTA TCA TCC CAA GA - #A GAG TTT TTG CAG TTG     5172     Cys Met Ile Gly His Cys Leu Ser Ser Gln Gl - #u Glu Phe Leu Gln Leu     #              16605     - GCT GGA AAA TGG GAC CTG GGA AAT TTG CTT CT - #C TTC AAC GGA GCT AAG     5220     Ala Gly Lys Trp Asp Leu Gly Asn Leu Leu Le - #u Phe Asn Gly Ala Lys     #          16750     - GTT GGT TCA CAA GAG GCC TTT TAT CTG TAT GC - #T TGT GGA CCC AAC CAT     5268     Val Gly Ser Gln Glu Ala Phe Tyr Leu Tyr Al - #a Cys Gly Pro Asn His     #      16905     - ACA TCT GTA ATG CCA TGT AAG TAT GGC AAG CC - #A GTC AAT GAC TAC TCC     5316     Thr Ser Val Met Pro Cys Lys Tyr Gly Lys Pr - #o Val Asn Asp Tyr Ser     #  17050     - AAA TAT ATT AAT AAA GAA ATT TTG CGA TGT GA - #A CAA ATC AGA GAA CTT     5364     Lys Tyr Ile Asn Lys Glu Ile Leu Arg Cys Gl - #u Gln Ile Arg Glu Leu     #               17251715 - #                1720     - TTT ATG ACC AAG AAA GAT GTG GAT ATT GGT CT - #C TTA ATT GAA AGT CTT     5412     Phe Met Thr Lys Lys Asp Val Asp Ile Gly Le - #u Leu Ile Glu Ser Leu     #              17405     - TCA GTT GTT TAT ACA ACT TAC TGT CCT GCT CA - #G TAT ACC ATC TAT GAA     5460     Ser Val Val Tyr Thr Thr Tyr Cys Pro Ala Gl - #n Tyr Thr Ile Tyr Glu     #          17550     - CCA GTG ATT AGA CTT AAA GGT CAA ATG AAA AC - #C CAA CTC TCT CAA AGA     5508     Pro Val Ile Arg Leu Lys Gly Gln Met Lys Th - #r Gln Leu Ser Gln Arg     #      17705     - CCC TTC AGC TCA AAA GAA GTT CAG AGC ATC TT - #A TTA GAA CCT CAT CAT     5556     Pro Phe Ser Ser Lys Glu Val Gln Ser Ile Le - #u Leu Glu Pro His His     #  17850     - CTA AAA AAT CTC CAA CCT ACT GAA TAT AAA AC - #T ATT CAA GGC ATT CTG     5604     Leu Lys Asn Leu Gln Pro Thr Glu Tyr Lys Th - #r Ile Gln Gly Ile Leu     #               18051795 - #                1800     - CAC GAA ATT GGT GGA ACT GGC ATA TTT GTT TT - #T CTC TTT GCC AGG GTT     5652     His Glu Ile Gly Gly Thr Gly Ile Phe Val Ph - #e Leu Phe Ala Arg Val     #              18205     - GTT GAA CTC AGT AGC TGT GAA GAA ACT CAA GC - #A TTA GCA CTG CGA GTT     5700     Val Glu Leu Ser Ser Cys Glu Glu Thr Gln Al - #a Leu Ala Leu Arg Val     #          18350     - ATA CTC TCA TTA ATT AAA TAC AAC CAA CAA AG - #A GTA CAT GAA TTA GAA     5748     Ile Leu Ser Leu Ile Lys Tyr Asn Gln Gln Ar - #g Val His Glu Leu Glu     #      18505     - AAT TGT AAT GGA CTT TCT ATG ATT CAT CAG GT - #G TTG ATC AAA CAA AAA     5796     Asn Cys Asn Gly Leu Ser Met Ile His Gln Va - #l Leu Ile Lys Gln Lys     #  18650     - TGC ATT GTT GGG TTT TAC ATT TTG AAG ACC CT - #T CTT GAA GGA TGC TGT     5844     Cys Ile Val Gly Phe Tyr Ile Leu Lys Thr Le - #u Leu Glu Gly Cys Cys     #               18851875 - #                1880     - GGT GAA GAT ATT ATT TAT ATG AAT GAG AAT GG - #A GAG TTT AAG TTG GAT     5892     Gly Glu Asp Ile Ile Tyr Met Asn Glu Asn Gl - #y Glu Phe Lys Leu Asp     #              19005     - GTA GAC TCT AAT GCT ATA ATC CAA GAT GTT AA - #G CTG TTA GAG GAA CTA     5940     Val Asp Ser Asn Ala Ile Ile Gln Asp Val Ly - #s Leu Leu Glu Glu Leu     #          19150     - TTG CTT GAC TGG AAG ATA TGG AGT AAA GCA GA - #G CAA GGT GTT TGG GAA     5988     Leu Leu Asp Trp Lys Ile Trp Ser Lys Ala Gl - #u Gln Gly Val Trp Glu     #      19305     - ACT TTG CTA GCA GCT CTA GAA GTC CTC ATC AG - #A GCA GAT CAC CAC CAG     6036     Thr Leu Leu Ala Ala Leu Glu Val Leu Ile Ar - #g Ala Asp His His Gln     #  19450     - CAG ATG TTT AAT ATT AAG CAG TTA TTG AAA GC - #T CAA GTG GTT CAT CAC     6084     Gln Met Phe Asn Ile Lys Gln Leu Leu Lys Al - #a Gln Val Val His His     #               19651955 - #                1960     - TTT CTA CTG ACT TGT CAG GTT TTG CAG GAA TA - #C AAA GAG GGG CAA CTC     6132     Phe Leu Leu Thr Cys Gln Val Leu Gln Glu Ty - #r Lys Glu Gly Gln Leu     #              19805     - ACA CCC ATG CCC CGA GAG GTT TGT AGA TCA TT - #T GTG AAA ATT ATA GCA     6180     Thr Pro Met Pro Arg Glu Val Cys Arg Ser Ph - #e Val Lys Ile Ile Ala     #          19950     - GAA GTC CTT GGA TCT CCT CCA GAT TTG GAA TT - #A TTG ACA ATT ATC TTC     6228     Glu Val Leu Gly Ser Pro Pro Asp Leu Glu Le - #u Leu Thr Ile Ile Phe     #      20105     - AAT TTC CTT TTA GCA GTT CAC CCT CCT ACT AA - #T ACT TAC GTT TGT CAC     6276     Asn Phe Leu Leu Ala Val His Pro Pro Thr As - #n Thr Tyr Val Cys His     #  20250     - AAT CCC ACG AAC TTC TAC TTT TCT TTG CAC AT - #A GAT GGC AAG ATC TTT     6324     Asn Pro Thr Asn Phe Tyr Phe Ser Leu His Il - #e Asp Gly Lys Ile Phe     #               20452035 - #                2040     - CAG GAG AAA GTG CGG TCA ATC ATG TAC CTG AG - #G CAT TCC AGC AGT GGA     6372     Gln Glu Lys Val Arg Ser Ile Met Tyr Leu Ar - #g His Ser Ser Ser Gly     #              20605     - GGA AGG TCC CTT ATG AGC CCT GGA TTT ATG GT - #A ATA AGC CCA TCT GGT     6420     Gly Arg Ser Leu Met Ser Pro Gly Phe Met Va - #l Ile Ser Pro Ser Gly     #          20750     - TTT ACT GCT TCA CCA TAT GAA GGA GAG AAT TC - #C TCT AAT ATT ATT CCA     6468     Phe Thr Ala Ser Pro Tyr Glu Gly Glu Asn Se - #r Ser Asn Ile Ile Pro     #      20905     - CAA CAG ATG GCC GCC CAT ATG CTG CGT TCT AG - #A AGC CTA CCA GCA TTC     6516     Gln Gln Met Ala Ala His Met Leu Arg Ser Ar - #g Ser Leu Pro Ala Phe     #  21050     - CCT ACT TCT TCA CTA CTA ACG CAA TCA CAA AA - #A CTG ACT GGA AGT TTG     6564     Pro Thr Ser Ser Leu Leu Thr Gln Ser Gln Ly - #s Leu Thr Gly Ser Leu     #               21252115 - #                2120     - GGT TGT AGT ATC GAC AGG TTA CAA AAT ATT GC - #A GAT ACT TAT GTT GCC     6612     Gly Cys Ser Ile Asp Arg Leu Gln Asn Ile Al - #a Asp Thr Tyr Val Ala     #              21405     - ACC CAA TCA AAG AAA CAA AAT TCT TTG GGG AG - #T TCC GAC ACA CTG AAA     6660     Thr Gln Ser Lys Lys Gln Asn Ser Leu Gly Se - #r Ser Asp Thr Leu Lys     #          21550     - AAA GGC AAA GAG GAC GCA TTC ATC AGT AGC TG - #T GAG TCT GCA AAA ACT     6708     Lys Gly Lys Glu Asp Ala Phe Ile Ser Ser Cy - #s Glu Ser Ala Lys Thr     #      21705     - GTT TGT GAA ATG GAA GCT GTC CTC TCA GCC CA - #G GTC TCT GTC AGT GAT     6756     Val Cys Glu Met Glu Ala Val Leu Ser Ala Gl - #n Val Ser Val Ser Asp     #  21850     - GTC CCA AAG GGA GTG CTG GGA TTT CCA GTG GT - #C AAA GCA GAT CAT AAA     6804     Val Pro Lys Gly Val Leu Gly Phe Pro Val Va - #l Lys Ala Asp His Lys     #               22052195 - #                2200     - CAG TTG GGA GCA GAA CCC AGG TCA GAA GAT GA - #C AGT CCT GGG GAT GAG     6852     Gln Leu Gly Ala Glu Pro Arg Ser Glu Asp As - #p Ser Pro Gly Asp Glu     #              22205     - TCC TGC CCA CGC CGA CCT GAT TAC CTA AAG GG - #A TTG GCC TCC TTC CAG     6900     Ser Cys Pro Arg Arg Pro Asp Tyr Leu Lys Gl - #y Leu Ala Ser Phe Gln     #          22350     - CGA AGC CAC AGC ACT ATT GCA AGC CTT GGG CT - #A GCT TTT CCT TCA CAG     6948     Arg Ser His Ser Thr Ile Ala Ser Leu Gly Le - #u Ala Phe Pro Ser Gln     #      22505     - AAC GGA TCT GCA GCT GTT GGC CGT TGG CCA AG - #T CTT GTT GAT AGA AAC     6996     Asn Gly Ser Ala Ala Val Gly Arg Trp Pro Se - #r Leu Val Asp Arg Asn     #  22650     - ACT GAT GAT TGG GAA AAC TTT GCC TAT TCT CT - #T GGT TAT GAG CCA AAT     7044     Thr Asp Asp Trp Glu Asn Phe Ala Tyr Ser Le - #u Gly Tyr Glu Pro Asn     #               22852275 - #                2280     - TAC AAC CGA ACT GCA AGT GCT CAC AGT GTA AC - #T GAA GAC TGT TTG GTA     7092     Tyr Asn Arg Thr Ala Ser Ala His Ser Val Th - #r Glu Asp Cys Leu Val     #              23005     - CCT ATA TGC TGT GGA TTA TAT GAA CTC CTA AG - #T GGG GTT CTT CTT ATC     7140     Pro Ile Cys Cys Gly Leu Tyr Glu Leu Leu Se - #r Gly Val Leu Leu Ile     #          23150     - CTG CCT GAT GTT TTG CTT GAA GAT GTG ATG GA - #C AAG CTT ATT CAA GCA     7188     Leu Pro Asp Val Leu Leu Glu Asp Val Met As - #p Lys Leu Ile Gln Ala     #      23305     - GAT ACA CTT TTG GTC CTC GTT AAC CAC CCA TC - #A CCA GCT ATA CAA CAA     7236     Asp Thr Leu Leu Val Leu Val Asn His Pro Se - #r Pro Ala Ile Gln Gln     #  23450     - GGT GTT ATT AAA CTA TTA GAT GCA TAT TTT GC - #T AGA GCA TCT AAG GAA     7284     Gly Val Ile Lys Leu Leu Asp Ala Tyr Phe Al - #a Arg Ala Ser Lys Glu     #               23652355 - #                2360     - CAA AAA GAT AAA TTT CTG AAG AAT CGT GGA TT - #T TCC TTG CTA GCC AAC     7332     Gln Lys Asp Lys Phe Leu Lys Asn Arg Gly Ph - #e Ser Leu Leu Ala Asn     #              23805     - CAG TTG TAT CTT CAT CGA GGA ACT CAA GAA TT - #G TTA GAA TGC TTC ATC     7380     Gln Leu Tyr Leu His Arg Gly Thr Gln Glu Le - #u Leu Glu Cys Phe Ile     #          23950     - GAA ATG TTC TTT GGT CGA CAT ATT GGC CTT GA - #T GAA GAA TTT GAT CTG     7428     Glu Met Phe Phe Gly Arg His Ile Gly Leu As - #p Glu Glu Phe Asp Leu     #      24105     - GAA GAT GTG AGA AAC ATG GGA TTG TTT CAG AA - #G TGG TCT GTC ATT CCT     7476     Glu Asp Val Arg Asn Met Gly Leu Phe Gln Ly - #s Trp Ser Val Ile Pro     #  24250     - ATT CTG GGA CTA ATA GAG ACC TCT CTA TAT GA - #C AAC ATA CTC TTG CAT     7524     Ile Leu Gly Leu Ile Glu Thr Ser Leu Tyr As - #p Asn Ile Leu Leu His     #               24452435 - #                2440     - AAT GCT CTT TTA CTT CTT CTC CAA ATT TTA AA - #T TCT TGT TCT AAG GTA     7572     Asn Ala Leu Leu Leu Leu Leu Gln Ile Leu As - #n Ser Cys Ser Lys Val     #              24605     - GCA GAT ATG TTG CTG GAT AAT GGT CTA CTC TA - #T GTG TTA TGT AAT ACA     7620     Ala Asp Met Leu Leu Asp Asn Gly Leu Leu Ty - #r Val Leu Cys Asn Thr     #          24750     - GTA GCA GCC CTG AAT GGA TTA GAA AAG AAC AT - #T CCC ATG AGT GAA TAT     7668     Val Ala Ala Leu Asn Gly Leu Glu Lys Asn Il - #e Pro Met Ser Glu Tyr     #      24905     - AAA TTG CTT GCT TGT GAT ATA CAG CAA CTT TT - #C ATA GCA GTT ACA ATT     7716     Lys Leu Leu Ala Cys Asp Ile Gln Gln Leu Ph - #e Ile Ala Val Thr Ile     #  25050     - CAT GCT TGC AGT TCC TCA GGC TCA CAA TAT TT - #T AGG GTT ATT GAA GAC     7764     His Ala Cys Ser Ser Ser Gly Ser Gln Tyr Ph - #e Arg Val Ile Glu Asp     #               25252515 - #                2520     - CTT ATT GTA ATG CTT GGA TAT CTT CAA AAT AG - #C AAA AAC AAG AGG ACA     7812     Leu Ile Val Met Leu Gly Tyr Leu Gln Asn Se - #r Lys Asn Lys Arg Thr     #              25405     - CAA AAT ATG GCT GTT GCA CTA CAG CTT AGA GT - #T CTC CAG GCT GCT ATG     7860     Gln Asn Met Ala Val Ala Leu Gln Leu Arg Va - #l Leu Gln Ala Ala Met     #          25550     - GAA TTT ATA AGG ACC ACC GCA AAT CAT GAC TC - #T GAA AAC CTC ACA GAT     7908     Glu Phe Ile Arg Thr Thr Ala Asn His Asp Se - #r Glu Asn Leu Thr Asp     #      25705     - TCA CTC CAG TCA CCT TCT GCT CCC CAT CAT GC - #A GTA GTT CAA AAG CGG     7956     Ser Leu Gln Ser Pro Ser Ala Pro His His Al - #a Val Val Gln Lys Arg     #  25850     - AAA AGC ATT GCT GGT CCT CGA AAA TTT CCC CT - #T GCT CAA ACT GAA TCG     8004     Lys Ser Ile Ala Gly Pro Arg Lys Phe Pro Le - #u Ala Gln Thr Glu Ser     #               26052595 - #                2600     - CTT CTG ATG AAA ATG CGT TCA GTG GCA AAT GA - #T GAG CTT CAT GTG ATG     8052     Leu Leu Met Lys Met Arg Ser Val Ala Asn As - #p Glu Leu His Val Met     #              26205     - ATG CAA CGG AGA ATG AGC CAA GAG AAC CCT AG - #C CAA GCA ACT GAA ACG     8100     Met Gln Arg Arg Met Ser Gln Glu Asn Pro Se - #r Gln Ala Thr Glu Thr     #          26350     - GAA CTT GCG CAG AGA CTA CAG AGG CTC ACT GT - #T TTA GCA GTC AAC AGG     8148     Glu Leu Ala Gln Arg Leu Gln Arg Leu Thr Va - #l Leu Ala Val Asn Arg     #      26505     - ATT ATT TAT CAA GAA TTT AAT TCA GAC ATT AT - #T GAC ATT TTG AGA ACT     8196     Ile Ile Tyr Gln Glu Phe Asn Ser Asp Ile Il - #e Asp Ile Leu Arg Thr     #  26650     - CCA GAA AAT GTA ACT CAA AGC AAG ACC TCA GT - #T TTC CAG ACC GAA ATT     8244     Pro Glu Asn Val Thr Gln Ser Lys Thr Ser Va - #l Phe Gln Thr Glu Ile     #               26852675 - #                2680     - TCT GAG GAA AAT ATT CAT CAT GAA CAG TCT TC - #T GTT TTC AAT CCA TTT     8292     Ser Glu Glu Asn Ile His His Glu Gln Ser Se - #r Val Phe Asn Pro Phe     #              27005     - CAG AAA GAA ATT TTT ACA TAT CTG GTA GAA GG - #A TTC AAA GTA TCT ATT     8340     Gln Lys Glu Ile Phe Thr Tyr Leu Val Glu Gl - #y Phe Lys Val Ser Ile     #          27150     - GGT TCA AGT AAA GCC AGT GGT TCC AAG CAG CA - #A TGG ACT AAA ATT CTG     8388     Gly Ser Ser Lys Ala Ser Gly Ser Lys Gln Gl - #n Trp Thr Lys Ile Leu     #      27305     - TGG TCT TGT AAG GAG ACC TTC CGA ATG CAG CT - #T GGG AGA CTA CTA GTG     8436     Trp Ser Cys Lys Glu Thr Phe Arg Met Gln Le - #u Gly Arg Leu Leu Val     #  27450     - CAT ATT TTG TCG CCA GCC CAC GCT GCA CAA GA - #G AGA AAG CAA ATT TTT     8484     His Ile Leu Ser Pro Ala His Ala Ala Gln Gl - #u Arg Lys Gln Ile Phe     #               27652755 - #                2760     - GAA ATA GTT CAT GAA CCA AAT CAT CAG GAA AT - #A CTA CGA GAC TGT CTC     8532     Glu Ile Val His Glu Pro Asn His Gln Glu Il - #e Leu Arg Asp Cys Leu     #              27805     - AGC CCA TCC CTA CAA CAT GGA GCC AAG TTA GT - #T TTG TAT TTG TCA GAG     8580     Ser Pro Ser Leu Gln His Gly Ala Lys Leu Va - #l Leu Tyr Leu Ser Glu     #          27950     - TTG ATA CAT AAT CAC CAA GGT GAA TTG ACT GA - #A GAA GAG CTA GGC ACA     8628     Leu Ile His Asn His Gln Gly Glu Leu Thr Gl - #u Glu Glu Leu Gly Thr     #      28105     - GCA GAA CTG CTT ATG AAT GCT TTG AAG TTA TG - #T GGT CAC AAG TGC ATC     8676     Ala Glu Leu Leu Met Asn Ala Leu Lys Leu Cy - #s Gly His Lys Cys Ile     #  28250     - CCT CCC AGT GCA TCA ACA AAA GCA GAC CTT AT - #T AAA ATG ATC AAA GAG     8724     Pro Pro Ser Ala Ser Thr Lys Ala Asp Leu Il - #e Lys Met Ile Lys Glu     #               28452835 - #                2840     - GAA CAA AAG AAA TAT GAA ACT GAA GAA GGA GT - #G AAT AAA GCT GCT TGG     8772     Glu Gln Lys Lys Tyr Glu Thr Glu Glu Gly Va - #l Asn Lys Ala Ala Trp     #              28605     - CAG AAA ACA GTT AAC AAT AAT CAA CAA AGT CT - #C TTT CAG CGT CTG GAT     8820     Gln Lys Thr Val Asn Asn Asn Gln Gln Ser Le - #u Phe Gln Arg Leu Asp     #          28750     - TCA AAA TCA AAG GAT ATA TCT AAA ATA GCT GC - #A GAT ATC ACC CAG GCA     8868     Ser Lys Ser Lys Asp Ile Ser Lys Ile Ala Al - #a Asp Ile Thr Gln Ala     #      28905     - GTG TCT CTC TCC CAA GGA AAT GAG AGA AAA AA - #G GTG ATC CAG CAT ATT     8916     Val Ser Leu Ser Gln Gly Asn Glu Arg Lys Ly - #s Val Ile Gln His Ile     #  29050     - AGA GGA ATG TAT AAA GTA GAT TTG AGT GCC AG - #C AGA CAT TGG CAG GAA     8964     Arg Gly Met Tyr Lys Val Asp Leu Ser Ala Se - #r Arg His Trp Gln Glu     #               29252915 - #                2920     - CTT ATT CAG CAG CTG ACA CAT GAT AGA GCA GT - #A TGG TAT GAC CCC ATC     9012     Leu Ile Gln Gln Leu Thr His Asp Arg Ala Va - #l Trp Tyr Asp Pro Ile     #              29405     - TAC TAT CCA ACC TCA TGG CAG TTG GAT CCA AC - #A GAA GGG CCA AAT CGA     9060     Tyr Tyr Pro Thr Ser Trp Gln Leu Asp Pro Th - #r Glu Gly Pro Asn Arg     #          29550     - GAG AGG AGA CGT TTA CAG AGA TGT TAT TTA AC - #T ATT CCA AAT AAG TAT     9108     Glu Arg Arg Arg Leu Gln Arg Cys Tyr Leu Th - #r Ile Pro Asn Lys Tyr     #      29705     - CTC CTT AGG GAT AGA CAG AAA TCA GAA GAT GT - #T GTC AAA CCA CCA CTC     9156     Leu Leu Arg Asp Arg Gln Lys Ser Glu Asp Va - #l Val Lys Pro Pro Leu     #  29850     - TCT TAC CTG TTT GAA GAC AAA ACT CAT TCT TC - #T TTC TCT TCT ACT GTC     9204     Ser Tyr Leu Phe Glu Asp Lys Thr His Ser Se - #r Phe Ser Ser Thr Val     #               30052995 - #                3000     - AAA GAC AAA GCT GCA AGT GAA TCT ATA AGA GT - #G AAT CGA AGA TGC ATC     9252     Lys Asp Lys Ala Ala Ser Glu Ser Ile Arg Va - #l Asn Arg Arg Cys Ile     #              30205     - AGT GTT GCA CCA TCT AGA GAG ACA GCT GGT GA - #A TTG TTA CTA GGT AAA     9300     Ser Val Ala Pro Ser Arg Glu Thr Ala Gly Gl - #u Leu Leu Leu Gly Lys     #          30350     - TGT GGA ATG TAT TTT GTG GAA GAT AAT GCT TC - #T GAT ACA GTT GAA AGT     9348     Cys Gly Met Tyr Phe Val Glu Asp Asn Ala Se - #r Asp Thr Val Glu Ser     #      30505     - TCG AGC CTT CAG GGA GAG TTG GAA CCA GCA TC - #A TTT TCC TGG ACA TAT     9396     Ser Ser Leu Gln Gly Glu Leu Glu Pro Ala Se - #r Phe Ser Trp Thr Tyr     #  30650     - GAA GAA ATT AAA GAA GTT CAC AAG CGT TGG TG - #G CAA TTG AGA GAT AAT     9444     Glu Glu Ile Lys Glu Val His Lys Arg Trp Tr - #p Gln Leu Arg Asp Asn     #               30853075 - #                3080     - GCT GTA GAA ATC TTT CTA ACA AAT GGC AGA AC - #A CTC CTG TTG GCA TTT     9492     Ala Val Glu Ile Phe Leu Thr Asn Gly Arg Th - #r Leu Leu Leu Ala Phe     #              31005     - GAT AAC ACC AAG GTT CGT GAT GAT GTA TAC CA - #C AAT ATA CTC ACA AAT     9540     Asp Asn Thr Lys Val Arg Asp Asp Val Tyr Hi - #s Asn Ile Leu Thr Asn     #          31150     - AAC CTC CCT AAT CTT CTG GAA TAT GGT AAC AT - #C ACC GCT CTG ACA AAT     9588     Asn Leu Pro Asn Leu Leu Glu Tyr Gly Asn Il - #e Thr Ala Leu Thr Asn     #      31305     - TTA TGG TAT ACT GGG CAA ATT ACT AAT TTT GA - #A TAT TTG ACT CAC TTA     9636     Leu Trp Tyr Thr Gly Gln Ile Thr Asn Phe Gl - #u Tyr Leu Thr His Leu     #  31450     - AAC AAA CAT GCT GGC CGA TCC TTC AAT GAT CT - #C ATG CAG TAT CCT GTG     9684     Asn Lys His Ala Gly Arg Ser Phe Asn Asp Le - #u Met Gln Tyr Pro Val     #               31653155 - #                3160     - TTC CCA TTT ATA CTT GCT GAC TAC GTT AGT GA - #G ACA CTT GAC CTC AAT     9732     Phe Pro Phe Ile Leu Ala Asp Tyr Val Ser Gl - #u Thr Leu Asp Leu Asn     #              31805     - GAT CTG TTG ATA TAC AGA AAT CTC TCT AAA CC - #T ATA GCT GTT CAG TAT     9780     Asp Leu Leu Ile Tyr Arg Asn Leu Ser Lys Pr - #o Ile Ala Val Gln Tyr     #          31950     - AAA GAA AAA GAA GAT CGT TAT GTG GAC ACA TA - #C AAG TAC TTG GAG GAA     9828     Lys Glu Lys Glu Asp Arg Tyr Val Asp Thr Ty - #r Lys Tyr Leu Glu Glu     #      32105     - GAG TAC CGC AAA GGA GCC AGA GAA GAT GAC CC - #C ATG CCT CCC GTG CAG     9876     Glu Tyr Arg Lys Gly Ala Arg Glu Asp Asp Pr - #o Met Pro Pro Val Gln     #  32250     - CCC TAT CAC TAT GGC TCC CAC TAT TCC AAT AG - #C GGC ACT GTG CTT CAC     9924     Pro Tyr His Tyr Gly Ser His Tyr Ser Asn Se - #r Gly Thr Val Leu His     #               32453235 - #                3240     - TTC CTG GTC AGG ATG CCT CCT TTC ACT AAA AT - #G TTT TTA GCC TAT CAA     9972     Phe Leu Val Arg Met Pro Pro Phe Thr Lys Me - #t Phe Leu Ala Tyr Gln     #              32605     - GAT CAA AGT TTT GAC ATT CCA GAC AGA ACT TT - #T CAT TCT ACA AAT ACA     10020     Asp Gln Ser Phe Asp Ile Pro Asp Arg Thr Ph - #e His Ser Thr Asn Thr     #          32750     - ACT TGG CGA CTC TCA TCT TTT GAA TCT ATG AC - #T GAT GTG AAA GAA CTT     10068     Thr Trp Arg Leu Ser Ser Phe Glu Ser Met Th - #r Asp Val Lys Glu Leu     #      32905     - ATC CCA GAG TTT TTC TAT CTT CCA GAG TTC CT - #A GTT AAC CGT GAA GGT     10116     Ile Pro Glu Phe Phe Tyr Leu Pro Glu Phe Le - #u Val Asn Arg Glu Gly     #  33050     - TTT GAT TTT GGT GTG CGT CAG AAT GGT GAA CG - #G GTT AAT CAC GTC AAC     10164     Phe Asp Phe Gly Val Arg Gln Asn Gly Glu Ar - #g Val Asn His Val Asn     #               33253315 - #                3320     - CTT CCC CCT TGG GCG CGT AAT GAT CCT CGT CT - #T TTT ATC CTC ATC CAT     10212     Leu Pro Pro Trp Ala Arg Asn Asp Pro Arg Le - #u Phe Ile Leu Ile His     #              33405     - CGG CAG GCT CTA GAG TCT GAC TAC GTG TCG CA - #G AAC ATC TGT CAG TGG     10260     Arg Gln Ala Leu Glu Ser Asp Tyr Val Ser Gl - #n Asn Ile Cys Gln Trp     #          33550     - ATT GAC TTG GTG TTT GGG TAT AAG CAA AAG GG - #G AAG GCT TCT GTT CAA     10308     Ile Asp Leu Val Phe Gly Tyr Lys Gln Lys Gl - #y Lys Ala Ser Val Gln     #      33705     - GCG ATC AAT GTT TTT CAT CCT GCT ACA TAT TT - #T GGA ATG GAT GTC TCT     10356     Ala Ile Asn Val Phe His Pro Ala Thr Tyr Ph - #e Gly Met Asp Val Ser     #  33850     - GCA GTT GAA GAT CCA GTT CAG AGA CGA GCG CT - #A GAA ACC ATG ATA AAA     10404     Ala Val Glu Asp Pro Val Gln Arg Arg Ala Le - #u Glu Thr Met Ile Lys     #               34053395 - #                3400     - ACC TAC GGG CAG ACT CCC CGT CAG CTG TTC CA - #C ATG GCC CAT GTG AGC     10452     Thr Tyr Gly Gln Thr Pro Arg Gln Leu Phe Hi - #s Met Ala His Val Ser     #              34205     - AGA CCT GGA GCC AAG CTC AAT ATT GAA GGA GA - #G CTT CCA GCT GCT GTG     10500     Arg Pro Gly Ala Lys Leu Asn Ile Glu Gly Gl - #u Leu Pro Ala Ala Val     #          34350     - GGG TTG CTA GTG CAG TTT GCT TTC AGG GAG AC - #C CGA GAA CAG GTC AAA     10548     Gly Leu Leu Val Gln Phe Ala Phe Arg Glu Th - #r Arg Glu Gln Val Lys     #      34505     - GAA ATC ACC TAT CCG AGT CCT TTG TCA TGG AT - #A AAA GGC TTG AAA TGG     10596     Glu Ile Thr Tyr Pro Ser Pro Leu Ser Trp Il - #e Lys Gly Leu Lys Trp     #  34650     - GGG GAA TAC GTG GGT TCC CCC AGT GCT CCA GT - #A CCT GTG GTC TGC TTC     10644     Gly Glu Tyr Val Gly Ser Pro Ser Ala Pro Va - #l Pro Val Val Cys Phe     #               34853475 - #                3480     - AGC CAG CCC CAC GGA GAA AGA TTT GGC TCT CT - #C CAG GCT CTG CCC ACC     10692     Ser Gln Pro His Gly Glu Arg Phe Gly Ser Le - #u Gln Ala Leu Pro Thr     #              35005     - AGA GCA ATC TGT GGT TTG TCA CGG AAT TTC TG - #T CTT GTG ATG ACA TAT     10740     Arg Ala Ile Cys Gly Leu Ser Arg Asn Phe Cy - #s Leu Val Met Thr Tyr     #          35150     - AGC AAG GAA CAA GGT GTG AGA AGC ATG AAC AG - #T ACG GAC ATT CAG TGG     10788     Ser Lys Glu Gln Gly Val Arg Ser Met Asn Se - #r Thr Asp Ile Gln Trp     #      35305     - TCA GCC ATC CTG AGC TGG GGA TAT GCT GAT AA - #T ATT TTA AGG TTG AAG     10836     Ser Ala Ile Leu Ser Trp Gly Tyr Ala Asp As - #n Ile Leu Arg Leu Lys     #  35450     - AGT AAA CAA AGT GAG CCT CCA GTA AAC TTT AT - #T CAA AGT TCA CAA CAG     10884     Ser Lys Gln Ser Glu Pro Pro Val Asn Phe Il - #e Gln Ser Ser Gln Gln     #               35653555 - #                3560     - TAC CAG GTG ACT AGT TGT GCT TGG GTG CCT GA - #C AGT TGC CAG CTG TTT     10932     Tyr Gln Val Thr Ser Cys Ala Trp Val Pro As - #p Ser Cys Gln Leu Phe     #              35805     - ACT GGA AGC AAA TGC GGT GTC ATC ACA GCC TA - #C ACA AAC AGA TTT ACA     10980     Thr Gly Ser Lys Cys Gly Val Ile Thr Ala Ty - #r Thr Asn Arg Phe Thr     #          35950     - AGC AGC ACG CCA TCA GAA ATA GAA ATG GAG AC - #T CAA ATA CAT CTC TAT     11028     Ser Ser Thr Pro Ser Glu Ile Glu Met Glu Th - #r Gln Ile His Leu Tyr     #      36105     - GGT CAC ACA GAA GAG ATA ACC AGC TTA TTT GT - #T TGC AAA CCA TAC AGT     11076     Gly His Thr Glu Glu Ile Thr Ser Leu Phe Va - #l Cys Lys Pro Tyr Ser     #  36250     - ATA CTG ATA AGT GTG AGC AGA GAC GGA ACC TG - #C ATC ATA TGG GAT TTA     11124     Ile Leu Ile Ser Val Ser Arg Asp Gly Thr Cy - #s Ile Ile Trp Asp Leu     #               36453635 - #                3640     - AAC AGG TTA TGC TAT GTA CAA AGT CTG GCG GG - #A CAC AAA AGC CCT GTC     11172     Asn Arg Leu Cys Tyr Val Gln Ser Leu Ala Gl - #y His Lys Ser Pro Val     #              36605     - ACA GCT GTC TCT GCC AGT GAA ACC TCA GGT GA - #T ATT GCT ACT GTG TGT     11220     Thr Ala Val Ser Ala Ser Glu Thr Ser Gly As - #p Ile Ala Thr Val Cys     #          36750     - GAT TCA GCT GGC GGA GGC AGT GAC CTC AGA CT - #C TGG ACG GTG AAC GGG     11268     Asp Ser Ala Gly Gly Gly Ser Asp Leu Arg Le - #u Trp Thr Val Asn Gly     #      36905     - GAT CTC GTT GGA CAT GTC CAC TGC AGG GAG AT - #C ATC TGT TCC GTG GCT     11316     Asp Leu Val Gly His Val His Cys Arg Glu Il - #e Ile Cys Ser Val Ala     #  37050     - TTC TCC AAC CAG CCT GAG GGA GTA TCT ATC AA - #T GTA ATC GCT GGG GGA     11364     Phe Ser Asn Gln Pro Glu Gly Val Ser Ile As - #n Val Ile Ala Gly Gly     #               37253715 - #                3720     - TTA GAA AAT GGA ATT GTT AGG TTA TGG AGC AC - #A TGG GAC TTA AAG CCT     11412     Leu Glu Asn Gly Ile Val Arg Leu Trp Ser Th - #r Trp Asp Leu Lys Pro     #              37405     - GTG AGA GAA ATT ACA TTT CCC AAA TCA AAT AA - #G CCC ATC ATC AGC CTT     11460     Val Arg Glu Ile Thr Phe Pro Lys Ser Asn Ly - #s Pro Ile Ile Ser Leu     #          37550     - ACA TTT TCT TGT GAT GGC CAC CAT TTG TAC AC - #A GCA AAC AGT GAT GGG     11508     Thr Phe Ser Cys Asp Gly His His Leu Tyr Th - #r Ala Asn Ser Asp Gly     #      37705     - ACC GTG ATT GCC TGG TGT CGG AAG GAC CAG CA - #C CGC TTG AAA CAG CCA     11556     Thr Val Ile Ala Trp Cys Arg Lys Asp Gln Hi - #s Arg Leu Lys Gln Pro     #  37850     - ATG TTC TAT TCC TTC CTT AGC AGC TAT GCA GC - #C GGG TGAATGCGAA     11602     Met Phe Tyr Ser Phe Leu Ser Ser Tyr Ala Al - #a Gly     3790                3795 - #                3800     - TGAACTTCAT GTTCTCCAAA GCACTTTAAC TCCAAACTAG ATTTGTTGAC TT - #CACCAGTT     11662     - TTAGGAGGTT GAACCTAAAG AAATGGATGA CTGGACAAAC CATCCAAATA AT - #GATAAAGT     11722     - CTATTCATCT GCACAAAATT CTGAAGAGTC ACATGATCCT AAGAGGAAAG TT - #CTGTTCTA     11782     - TTTTAGTGAT AATCTGGAAG ATTGTGTCAA TATGCACTAG CCAACAAGTT TT - #AAGCCTCG     11842     - CATGGTACAT TAAAATGATA TTCTTAAAAT TTTTTCCCAC CAAGGTATTC CA - #AAGAAAAT     11902     - ATTAAGGTCT CCCCTTTTTC TATGATTCCA AAAGGACCAG TAGAATTTAA AT - #TGGTTGGT     11962     - TGATNGTTTA TATAAAACAC ACTAAAATTA TATTTTAAAA GTTTANTGCCN T - #GAAATACT     12022     - CCTCCCACCA CACACACATG CTCCAAAAGA GGAAAGAAAA AAAGATAATTT T - #TAGGACTT     12082     - GATAATTGCT TTCTTTGAGA AGCAAATTAT TCAGTAGGTG CCTCTGTACCA A - #ATATTTTA     12142     - TGGAATATCT AAATACTAAA ATAAACTATG AATGAATCTC AAAATTAGGCA G - #TTTTTGCC     12202     - TGCTTTCTTA GCTCAAAGGA GAACCAGAAT TTTTTTGACA GCCACAAACA AG - #AATACAGG     12262     - AGTTATCTTG GATTTCAGAC ACATTCTGTT TCTTCATAAA AATTTTACTT AA - #AATCTGTA     12322     - ACGCTAGATA TTGACTATCC TTAGTTGAGT CACTGAGGTT TAAACACAAT GG - #TAAGTCTT     12382     - AAAGTCTGCT ATTTACAGAG CATTGAATCT GTACCAATTT GCAATAGAAA GC - #CTTCAGTA     12442     - TGCAAGAAGT TTGCATGGGT ATTAAGAACA CAGCCTAAAT AAGGCATTTG AT - #CTAATCTG     12502     - CAGGAAGAAT TTTCTTCCCC AAAACAGAAT TATAAAAGCT TACTTTAAAC AG - #GAGGCAGA     12562     - ATAATTCTTT TAGGAAACCA TTTCATTCTG TTTCTACTAA CCTATACCAT CT - #GA     12616     - (2) INFORMATION FOR SEQ ID NO:10:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 3801 amino               (B) TYPE: amino acid               (D) TOPOLOGY: unknown     -     (ii) MOLECULE TYPE: protein     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:     - Met Ser Thr Asp Ser Asn Ser Leu Ala Arg Gl - #u Phe Leu Thr Asp Val     #                 15     - Asn Arg Leu Cys Asn Ala Val Val Gln Arg Va - #l Glu Ala Arg Glu Glu     #             30     - Glu Glu Glu Glu Thr His Met Ala Thr Leu Gl - #y Gln Tyr Leu Val His     #         45     - Gly Arg Gly Phe Leu Leu Leu Thr Lys Leu As - #n Ser Ile Ile Asp Gln     #     60     - Ala Leu Thr Cys Arg Glu Glu Leu Leu Thr Le - #u Leu Leu Ser Leu Leu     # 80     - Pro Leu Val Trp Lys Ile Pro Val Gln Glu Gl - #u Lys Ala Thr Asp Phe     #                 95     - Asn Leu Pro Leu Ser Ala Asp Ile Ile Leu Th - #r Lys Glu Lys Asn Ser     #           110     - Ser Ser Gln Arg Ser Thr Gln Glu Lys Leu Hi - #s Leu Glu Gly Ser Ala     #       125     - Leu Ser Ser Gln Val Ser Ala Lys Val Asn Va - #l Phe Arg Lys Ser Arg     #   140     - Arg Gln Arg Lys Ile Thr His Arg Tyr Ser Va - #l Arg Asp Ala Arg Lys     145                 1 - #50                 1 - #55                 1 -     #60     - Thr Gln Leu Ser Thr Ser Asp Ser Glu Ala As - #n Ser Asp Glu Lys Gly     #               175     - Ile Ala Met Asn Lys His Arg Arg Pro His Le - #u Leu His His Phe Leu     #           190     - Thr Ser Phe Pro Lys Gln Asp His Pro Lys Al - #a Lys Leu Asp Arg Leu     #       205     - Ala Thr Lys Glu Gln Thr Pro Pro Asp Ala Me - #t Ala Leu Glu Asn Ser     #   220     - Arg Glu Ile Ile Pro Arg Gln Gly Ser Asn Th - #r Asp Ile Leu Ser Glu     225                 2 - #30                 2 - #35                 2 -     #40     - Pro Ala Ala Leu Ser Val Ile Ser Asn Met As - #n Asn Ser Pro Phe Asp     #               255     - Leu Cys His Val Leu Leu Ser Leu Leu Glu Ly - #s Val Cys Lys Phe Asp     #           270     - Val Thr Leu Asn His Asn Ser Pro Leu Ala Al - #a Ser Val Val Pro Thr     #       285     - Leu Thr Glu Phe Leu Ala Gly Phe Gly Asp Cy - #s Cys Ser Leu Ser Asp     #   300     - Asn Leu Glu Ser Arg Val Val Ser Ala Gly Tr - #p Thr Glu Glu Pro Val     305                 3 - #10                 3 - #15                 3 -     #20     - Ala Leu Ile Gln Arg Met Leu Phe Arg Thr Va - #l Leu His Leu Leu Ser     #               335     - Val Asp Val Ser Thr Ala Glu Met Met Pro Gl - #u Asn Leu Arg Lys Asn     #           350     - Leu Thr Glu Leu Leu Arg Ala Ala Leu Lys Il - #e Arg Ile Cys Leu Glu     #       365     - Lys Gln Pro Asp Pro Phe Ala Pro Arg Gln Ly - #s Lys Thr Leu Gln Glu     #   380     - Val Gln Glu Asp Phe Val Phe Ser Lys Tyr Ar - #g His Arg Ala Leu Leu     385                 3 - #90                 3 - #95                 4 -     #00     - Leu Pro Glu Leu Leu Glu Gly Val Leu Gln Il - #e Leu Ile Cys Cys Leu     #               415     - Gln Ser Ala Ala Ser Asn Pro Phe Tyr Phe Se - #r Gln Ala Met Asp Leu     #           430     - Val Gln Glu Phe Ile Gln His His Gly Phe As - #n Leu Phe Glu Thr Ala     #       445     - Val Leu Gln Met Glu Trp Leu Val Leu Arg As - #p Gly Val Pro Pro Glu     #   460     - Ala Ser Glu His Leu Lys Ala Leu Ile Asn Se - #r Val Met Lys Ile Met     465                 4 - #70                 4 - #75                 4 -     #80     - Ser Thr Val Lys Lys Val Lys Ser Glu Gln Le - #u His His Ser Met Cys     #               495     - Thr Arg Lys Arg His Arg Arg Cys Glu Tyr Se - #r His Phe Met His His     #           510     - His Arg Asp Leu Ser Gly Leu Leu Val Ser Al - #a Phe Lys Asn Gln Val     #       525     - Ser Lys Asn Pro Phe Glu Glu Thr Ala Asp Gl - #y Asp Val Tyr Tyr Pro     #   540     - Glu Arg Cys Cys Cys Ile Ala Val Cys Ala Hi - #s Gln Cys Leu Arg Leu     545                 5 - #50                 5 - #55                 5 -     #60     - Leu Gln Gln Ala Ser Leu Ser Ser Thr Cys Va - #l Gln Ile Leu Ser Gly     #               575     - Val His Asn Ile Gly Ile Cys Cys Cys Met As - #p Pro Lys Ser Val Ile     #           590     - Ile Pro Leu Leu His Ala Phe Lys Leu Pro Al - #a Leu Lys Asn Phe Gln     #       605     - Gln His Ile Leu Asn Ile Leu Asn Lys Leu Il - #e Leu Asp Gln Leu Gly     #   620     - Gly Ala Glu Ile Ser Pro Lys Ile Lys Lys Al - #a Ala Cys Asn Ile Cys     625                 6 - #30                 6 - #35                 6 -     #40     - Thr Val Asp Ser Asp Gln Leu Ala Gln Leu Gl - #u Glu Thr Leu Gln Gly     #               655     - Asn Leu Cys Asp Ala Glu Leu Ser Ser Ser Le - #u Ser Ser Pro Ser Tyr     #           670     - Arg Phe Gln Gly Ile Leu Pro Ser Ser Gly Se - #r Glu Asp Leu Leu Trp     #       685     - Lys Trp Asp Ala Leu Lys Ala Tyr Gln Asn Ph - #e Val Phe Glu Glu Asp     #   700     - Arg Leu His Ser Ile Gln Ile Ala Asn His Il - #e Cys Asn Leu Ile Gln     705                 7 - #10                 7 - #15                 7 -     #20     - Lys Gly Asn Ile Val Val Gln Trp Lys Leu Ty - #r Asn Tyr Ile Phe Asn     #               735     - Pro Val Leu Gln Arg Gly Val Glu Leu Ala Hi - #s His Cys Gln His Leu     #           750     - Ser Val Thr Ser Ala Gln Ser His Val Cys Se - #r His His Asn Gln Cys     #       765     - Leu Pro Gln Asp Val Leu Gln Ile Tyr Val Ly - #s Thr Leu Pro Ile Leu     #   780     - Leu Lys Ser Arg Val Ile Arg Asp Leu Phe Le - #u Ser Cys Asn Gly Val     785                 7 - #90                 7 - #95                 8 -     #00     - Ser Gln Ile Ile Glu Leu Asn Cys Leu Asn Gl - #y Ile Arg Ser His Ser     #               815     - Leu Lys Ala Phe Glu Thr Leu Ile Ile Ser Le - #u Gly Glu Gln Gln Lys     #           830     - Asp Ala Ser Val Pro Asp Ile Asp Gly Ile As - #p Ile Glu Gln Lys Glu     #       845     - Leu Ser Ser Val His Val Gly Thr Ser Phe Hi - #s His Gln Gln Ala Tyr     #   860     - Ser Asp Ser Pro Gln Ser Leu Ser Lys Phe Ty - #r Ala Gly Leu Lys Glu     865                 8 - #70                 8 - #75                 8 -     #80     - Ala Tyr Pro Lys Arg Arg Lys Thr Val Asn Gl - #n Asp Val His Ile Asn     #               895     - Thr Ile Asn Leu Phe Leu Cys Val Ala Phe Le - #u Cys Val Ser Lys Glu     #           910     - Ala Glu Ser Asp Arg Glu Ser Ala Asn Asp Se - #r Glu Asp Thr Ser Gly     #       925     - Tyr Asp Ser Thr Ala Ser Glu Pro Leu Ser Hi - #s Met Leu Pro Cys Ile     #   940     - Ser Leu Glu Ser Leu Val Leu Pro Ser Pro Gl - #u His Met His Gln Ala     945                 9 - #50                 9 - #55                 9 -     #60     - Ala Asp Ile Trp Ser Met Cys Arg Trp Ile Ty - #r Met Leu Ser Ser Val     #               975     - Phe Gln Lys Gln Phe Tyr Arg Leu Gly Gly Ph - #e Arg Val Cys His Lys     #           990     - Leu Ile Phe Met Ile Ile Gln Lys Leu Phe Ar - #g Ser His Lys Glu Glu     #      10050     - Gln Gly Lys Lys Glu Gly Asp Thr Ser Val As - #n Glu Asn Gln Asp Leu     #  10205     - Asn Arg Ile Ser Gln Pro Lys Arg Thr Met Ly - #s Glu Asp Leu Leu Ser     #               10401030 - #                1035     - Leu Ala Ile Lys Ser Asp Pro Ile Pro Ser Gl - #u Leu Gly Ser Leu Lys     #              10550     - Lys Ser Ala Asp Ser Leu Gly Lys Leu Glu Le - #u Gln His Ile Ser Ser     #          10705     - Ile Asn Val Glu Glu Val Ser Ala Thr Glu Al - #a Ala Pro Glu Glu Ala     #      10850     - Lys Leu Phe Thr Ser Gln Glu Ser Glu Thr Se - #r Leu Gln Ser Ile Arg     #  11005     - Leu Leu Glu Ala Leu Leu Ala Ile Cys Leu Hi - #s Gly Ala Arg Thr Ser     #               11201110 - #                1115     - Gln Gln Lys Met Glu Leu Glu Leu Pro Asn Gl - #n Asn Leu Ser Val Glu     #              11350     - Ser Ile Leu Phe Glu Met Arg Asp His Leu Se - #r Gln Ser Lys Val Ile     #          11505     - Glu Thr Gln Leu Ala Lys Pro Leu Phe Asp Al - #a Leu Leu Arg Val Ala     #      11650     - Leu Gly Asn Tyr Ser Ala Asp Phe Glu His As - #n Asp Ala Met Thr Glu     #  11805     - Lys Ser His Gln Ser Ala Glu Glu Leu Ser Se - #r Gln Pro Gly Asp Phe     #               12001190 - #                1195     - Ser Glu Glu Ala Glu Asp Ser Gln Cys Cys Se - #r Phe Lys Leu Leu Val     #              12150     - Glu Glu Glu Gly Tyr Glu Ala Asp Ser Glu Se - #r Asn Pro Glu Asp Gly     #          12305     - Glu Thr Gln Asp Asp Gly Val Asp Leu Lys Se - #r Glu Thr Glu Gly Phe     #      12450     - Ser Ala Ser Ser Ser Pro Asn Asp Leu Leu Gl - #u Asn Leu Thr Gln Gly     #  12605     - Glu Ile Ile Tyr Pro Glu Ile Cys Met Leu Gl - #u Leu Asn Leu Leu Ser     #               12801270 - #                1275     - Ala Ser Lys Ala Lys Leu Asp Val Leu Ala Hi - #s Val Phe Glu Ser Phe     #              12950     - Leu Lys Ile Ile Arg Gln Lys Glu Lys Asn Va - #l Phe Leu Leu Met Gln     #          13105     - Gln Gly Thr Val Lys Asn Leu Leu Gly Gly Ph - #e Leu Ser Ile Leu Thr     #      13250     - Gln Asp Asp Ser Asp Phe Gln Ala Cys Gln Ar - #g Val Leu Val Asp Leu     #  13405     - Leu Val Ser Leu Met Ser Ser Arg Thr Cys Se - #r Glu Glu Leu Thr Leu     #               13601350 - #                1355     - Leu Leu Arg Ile Phe Leu Glu Lys Ser Pro Cy - #s Thr Lys Ile Leu Leu     #              13750     - Leu Gly Ile Leu Lys Ile Ile Glu Ser Asp Th - #r Thr Met Ser Pro Ser     #          13905     - Gln Tyr Leu Thr Phe Pro Leu Leu His Ala Pr - #o Asn Leu Ser Asn Gly     #      14050     - Val Ser Ser Gln Lys Tyr Pro Gly Ile Leu As - #n Ser Lys Ala Met Gly     #  14205     - Leu Leu Arg Arg Ala Arg Val Ser Arg Ser Ly - #s Lys Glu Ala Asp Arg     #               14401430 - #                1435     - Glu Ser Phe Pro His Arg Leu Leu Ser Ser Tr - #p His Ile Ala Pro Val     #              14550     - His Leu Pro Leu Leu Gly Gln Asn Cys Trp Pr - #o His Leu Ser Glu Gly     #          14705     - Phe Ser Val Ser Leu Trp Phe Asn Val Glu Cy - #s Ile His Glu Ala Glu     #      14850     - Ser Thr Thr Glu Lys Gly Lys Lys Ile Lys Ly - #s Arg Asn Lys Ser Leu     #  15005     - Ile Leu Pro Asp Ser Ser Phe Asp Gly Thr Gl - #u Ser Asp Arg Pro Glu     #               15201510 - #                1515     - Gly Ala Glu Tyr Ile Asn Pro Gly Glu Arg Le - #u Ile Glu Glu Gly Cys     #              15350     - Ile His Ile Ile Ser Leu Gly Ser Lys Ala Le - #u Met Ile Gln Val Trp     #          15505     - Ala Asp Pro His Asn Ala Thr Leu Ile Phe Ar - #g Val Cys Met Asp Ser     #      15650     - Asn Asp Asp Met Lys Ala Val Leu Leu Ala Gl - #n Val Glu Ser Gln Glu     #  15805     - Asn Ile Phe Leu Pro Ser Lys Trp Gln His Le - #u Val Leu Thr Tyr Leu     #               16001590 - #                1595     - Gln Gln Pro Gln Gly Lys Arg Arg Ile His Gl - #y Lys Ile Ser Ile Trp     #              16150     - Val Ser Gly Gln Arg Lys Pro Asp Val Thr Le - #u Asp Phe Met Leu Pro     #          16305     - Arg Lys Thr Ser Leu Ser Ser Asp Ser Asn Ly - #s Thr Phe Cys Met Ile     #      16450     - Gly His Cys Leu Ser Ser Gln Glu Glu Phe Le - #u Gln Leu Ala Gly Lys     #  16605     - Trp Asp Leu Gly Asn Leu Leu Leu Phe Asn Gl - #y Ala Lys Val Gly Ser     #               16801670 - #                1675     - Gln Glu Ala Phe Tyr Leu Tyr Ala Cys Gly Pr - #o Asn His Thr Ser Val     #              16950     - Met Pro Cys Lys Tyr Gly Lys Pro Val Asn As - #p Tyr Ser Lys Tyr Ile     #          17105     - Asn Lys Glu Ile Leu Arg Cys Glu Gln Ile Ar - #g Glu Leu Phe Met Thr     #      17250     - Lys Lys Asp Val Asp Ile Gly Leu Leu Ile Gl - #u Ser Leu Ser Val Val     #  17405     - Tyr Thr Thr Tyr Cys Pro Ala Gln Tyr Thr Il - #e Tyr Glu Pro Val Ile     #               17601750 - #                1755     - Arg Leu Lys Gly Gln Met Lys Thr Gln Leu Se - #r Gln Arg Pro Phe Ser     #              17750     - Ser Lys Glu Val Gln Ser Ile Leu Leu Glu Pr - #o His His Leu Lys Asn     #          17905     - Leu Gln Pro Thr Glu Tyr Lys Thr Ile Gln Gl - #y Ile Leu His Glu Ile     #      18050     - Gly Gly Thr Gly Ile Phe Val Phe Leu Phe Al - #a Arg Val Val Glu Leu     #  18205     - Ser Ser Cys Glu Glu Thr Gln Ala Leu Ala Le - #u Arg Val Ile Leu Ser     #               18401830 - #                1835     - Leu Ile Lys Tyr Asn Gln Gln Arg Val His Gl - #u Leu Glu Asn Cys Asn     #              18550     - Gly Leu Ser Met Ile His Gln Val Leu Ile Ly - #s Gln Lys Cys Ile Val     #          18705     - Gly Phe Tyr Ile Leu Lys Thr Leu Leu Glu Gl - #y Cys Cys Gly Glu Asp     #      18850     - Ile Ile Tyr Met Asn Glu Asn Gly Glu Phe Ly - #s Leu Asp Val Asp Ser     #  19005     - Asn Ala Ile Ile Gln Asp Val Lys Leu Leu Gl - #u Glu Leu Leu Leu Asp     #               19201910 - #                1915     - Trp Lys Ile Trp Ser Lys Ala Glu Gln Gly Va - #l Trp Glu Thr Leu Leu     #              19350     - Ala Ala Leu Glu Val Leu Ile Arg Ala Asp Hi - #s His Gln Gln Met Phe     #          19505     - Asn Ile Lys Gln Leu Leu Lys Ala Gln Val Va - #l His His Phe Leu Leu     #      19650     - Thr Cys Gln Val Leu Gln Glu Tyr Lys Glu Gl - #y Gln Leu Thr Pro Met     #  19805     - Pro Arg Glu Val Cys Arg Ser Phe Val Lys Il - #e Ile Ala Glu Val Leu     #               20001990 - #                1995     - Gly Ser Pro Pro Asp Leu Glu Leu Leu Thr Il - #e Ile Phe Asn Phe Leu     #              20150     - Leu Ala Val His Pro Pro Thr Asn Thr Tyr Va - #l Cys His Asn Pro Thr     #          20305     - Asn Phe Tyr Phe Ser Leu His Ile Asp Gly Ly - #s Ile Phe Gln Glu Lys     #      20450     - Val Arg Ser Ile Met Tyr Leu Arg His Ser Se - #r Ser Gly Gly Arg Ser     #  20605     - Leu Met Ser Pro Gly Phe Met Val Ile Ser Pr - #o Ser Gly Phe Thr Ala     #               20802070 - #                2075     - Ser Pro Tyr Glu Gly Glu Asn Ser Ser Asn Il - #e Ile Pro Gln Gln Met     #              20950     - Ala Ala His Met Leu Arg Ser Arg Ser Leu Pr - #o Ala Phe Pro Thr Ser     #          21105     - Ser Leu Leu Thr Gln Ser Gln Lys Leu Thr Gl - #y Ser Leu Gly Cys Ser     #      21250     - Ile Asp Arg Leu Gln Asn Ile Ala Asp Thr Ty - #r Val Ala Thr Gln Ser     #  21405     - Lys Lys Gln Asn Ser Leu Gly Ser Ser Asp Th - #r Leu Lys Lys Gly Lys     #               21602150 - #                2155     - Glu Asp Ala Phe Ile Ser Ser Cys Glu Ser Al - #a Lys Thr Val Cys Glu     #              21750     - Met Glu Ala Val Leu Ser Ala Gln Val Ser Va - #l Ser Asp Val Pro Lys     #          21905     - Gly Val Leu Gly Phe Pro Val Val Lys Ala As - #p His Lys Gln Leu Gly     #      22050     - Ala Glu Pro Arg Ser Glu Asp Asp Ser Pro Gl - #y Asp Glu Ser Cys Pro     #  22205     - Arg Arg Pro Asp Tyr Leu Lys Gly Leu Ala Se - #r Phe Gln Arg Ser His     #               22402230 - #                2235     - Ser Thr Ile Ala Ser Leu Gly Leu Ala Phe Pr - #o Ser Gln Asn Gly Ser     #              22550     - Ala Ala Val Gly Arg Trp Pro Ser Leu Val As - #p Arg Asn Thr Asp Asp     #          22705     - Trp Glu Asn Phe Ala Tyr Ser Leu Gly Tyr Gl - #u Pro Asn Tyr Asn Arg     #      22850     - Thr Ala Ser Ala His Ser Val Thr Glu Asp Cy - #s Leu Val Pro Ile Cys     #  23005     - Cys Gly Leu Tyr Glu Leu Leu Ser Gly Val Le - #u Leu Ile Leu Pro Asp     #               23202310 - #                2315     - Val Leu Leu Glu Asp Val Met Asp Lys Leu Il - #e Gln Ala Asp Thr Leu     #              23350     - Leu Val Leu Val Asn His Pro Ser Pro Ala Il - #e Gln Gln Gly Val Ile     #          23505     - Lys Leu Leu Asp Ala Tyr Phe Ala Arg Ala Se - #r Lys Glu Gln Lys Asp     #      23650     - Lys Phe Leu Lys Asn Arg Gly Phe Ser Leu Le - #u Ala Asn Gln Leu Tyr     #  23805     - Leu His Arg Gly Thr Gln Glu Leu Leu Glu Cy - #s Phe Ile Glu Met Phe     #               24002390 - #                2395     - Phe Gly Arg His Ile Gly Leu Asp Glu Glu Ph - #e Asp Leu Glu Asp Val     #              24150     - Arg Asn Met Gly Leu Phe Gln Lys Trp Ser Va - #l Ile Pro Ile Leu Gly     #          24305     - Leu Ile Glu Thr Ser Leu Tyr Asp Asn Ile Le - #u Leu His Asn Ala Leu     #      24450     - Leu Leu Leu Leu Gln Ile Leu Asn Ser Cys Se - #r Lys Val Ala Asp Met     #  24605     - Leu Leu Asp Asn Gly Leu Leu Tyr Val Leu Cy - #s Asn Thr Val Ala Ala     #               24802470 - #                2475     - Leu Asn Gly Leu Glu Lys Asn Ile Pro Met Se - #r Glu Tyr Lys Leu Leu     #              24950     - Ala Cys Asp Ile Gln Gln Leu Phe Ile Ala Va - #l Thr Ile His Ala Cys     #          25105     - Ser Ser Ser Gly Ser Gln Tyr Phe Arg Val Il - #e Glu Asp Leu Ile Val     #      25250     - Met Leu Gly Tyr Leu Gln Asn Ser Lys Asn Ly - #s Arg Thr Gln Asn Met     #  25405     - Ala Val Ala Leu Gln Leu Arg Val Leu Gln Al - #a Ala Met Glu Phe Ile     #               25602550 - #                2555     - Arg Thr Thr Ala Asn His Asp Ser Glu Asn Le - #u Thr Asp Ser Leu Gln     #              25750     - Ser Pro Ser Ala Pro His His Ala Val Val Gl - #n Lys Arg Lys Ser Ile     #          25905     - Ala Gly Pro Arg Lys Phe Pro Leu Ala Gln Th - #r Glu Ser Leu Leu Met     #      26050     - Lys Met Arg Ser Val Ala Asn Asp Glu Leu Hi - #s Val Met Met Gln Arg     #  26205     - Arg Met Ser Gln Glu Asn Pro Ser Gln Ala Th - #r Glu Thr Glu Leu Ala     #               26402630 - #                2635     - Gln Arg Leu Gln Arg Leu Thr Val Leu Ala Va - #l Asn Arg Ile Ile Tyr     #              26550     - Gln Glu Phe Asn Ser Asp Ile Ile Asp Ile Le - #u Arg Thr Pro Glu Asn     #          26705     - Val Thr Gln Ser Lys Thr Ser Val Phe Gln Th - #r Glu Ile Ser Glu Glu     #      26850     - Asn Ile His His Glu Gln Ser Ser Val Phe As - #n Pro Phe Gln Lys Glu     #  27005     - Ile Phe Thr Tyr Leu Val Glu Gly Phe Lys Va - #l Ser Ile Gly Ser Ser     #               27202710 - #                2715     - Lys Ala Ser Gly Ser Lys Gln Gln Trp Thr Ly - #s Ile Leu Trp Ser Cys     #              27350     - Lys Glu Thr Phe Arg Met Gln Leu Gly Arg Le - #u Leu Val His Ile Leu     #          27505     - Ser Pro Ala His Ala Ala Gln Glu Arg Lys Gl - #n Ile Phe Glu Ile Val     #      27650     - His Glu Pro Asn His Gln Glu Ile Leu Arg As - #p Cys Leu Ser Pro Ser     #  27805     - Leu Gln His Gly Ala Lys Leu Val Leu Tyr Le - #u Ser Glu Leu Ile His     #               28002790 - #                2795     - Asn His Gln Gly Glu Leu Thr Glu Glu Glu Le - #u Gly Thr Ala Glu Leu     #              28150     - Leu Met Asn Ala Leu Lys Leu Cys Gly His Ly - #s Cys Ile Pro Pro Ser     #          28305     - Ala Ser Thr Lys Ala Asp Leu Ile Lys Met Il - #e Lys Glu Glu Gln Lys     #      28450     - Lys Tyr Glu Thr Glu Glu Gly Val Asn Lys Al - #a Ala Trp Gln Lys Thr     #  28605     - Val Asn Asn Asn Gln Gln Ser Leu Phe Gln Ar - #g Leu Asp Ser Lys Ser     #               28802870 - #                2875     - Lys Asp Ile Ser Lys Ile Ala Ala Asp Ile Th - #r Gln Ala Val Ser Leu     #              28950     - Ser Gln Gly Asn Glu Arg Lys Lys Val Ile Gl - #n His Ile Arg Gly Met     #          29105     - Tyr Lys Val Asp Leu Ser Ala Ser Arg His Tr - #p Gln Glu Leu Ile Gln     #      29250     - Gln Leu Thr His Asp Arg Ala Val Trp Tyr As - #p Pro Ile Tyr Tyr Pro     #  29405     - Thr Ser Trp Gln Leu Asp Pro Thr Glu Gly Pr - #o Asn Arg Glu Arg Arg     #               29602950 - #                2955     - Arg Leu Gln Arg Cys Tyr Leu Thr Ile Pro As - #n Lys Tyr Leu Leu Arg     #              29750     - Asp Arg Gln Lys Ser Glu Asp Val Val Lys Pr - #o Pro Leu Ser Tyr Leu     #          29905     - Phe Glu Asp Lys Thr His Ser Ser Phe Ser Se - #r Thr Val Lys Asp Lys     #      30050     - Ala Ala Ser Glu Ser Ile Arg Val Asn Arg Ar - #g Cys Ile Ser Val Ala     #  30205     - Pro Ser Arg Glu Thr Ala Gly Glu Leu Leu Le - #u Gly Lys Cys Gly Met     #               30403030 - #                3035     - Tyr Phe Val Glu Asp Asn Ala Ser Asp Thr Va - #l Glu Ser Ser Ser Leu     #              30550     - Gln Gly Glu Leu Glu Pro Ala Ser Phe Ser Tr - #p Thr Tyr Glu Glu Ile     #          30705     - Lys Glu Val His Lys Arg Trp Trp Gln Leu Ar - #g Asp Asn Ala Val Glu     #      30850     - Ile Phe Leu Thr Asn Gly Arg Thr Leu Leu Le - #u Ala Phe Asp Asn Thr     #  31005     - Lys Val Arg Asp Asp Val Tyr His Asn Ile Le - #u Thr Asn Asn Leu Pro     #               31203110 - #                3115     - Asn Leu Leu Glu Tyr Gly Asn Ile Thr Ala Le - #u Thr Asn Leu Trp Tyr     #              31350     - Thr Gly Gln Ile Thr Asn Phe Glu Tyr Leu Th - #r His Leu Asn Lys His     #          31505     - Ala Gly Arg Ser Phe Asn Asp Leu Met Gln Ty - #r Pro Val Phe Pro Phe     #      31650     - Ile Leu Ala Asp Tyr Val Ser Glu Thr Leu As - #p Leu Asn Asp Leu Leu     #  31805     - Ile Tyr Arg Asn Leu Ser Lys Pro Ile Ala Va - #l Gln Tyr Lys Glu Lys     #               32003190 - #                3195     - Glu Asp Arg Tyr Val Asp Thr Tyr Lys Tyr Le - #u Glu Glu Glu Tyr Arg     #              32150     - Lys Gly Ala Arg Glu Asp Asp Pro Met Pro Pr - #o Val Gln Pro Tyr His     #          32305     - Tyr Gly Ser His Tyr Ser Asn Ser Gly Thr Va - #l Leu His Phe Leu Val     #      32450     - Arg Met Pro Pro Phe Thr Lys Met Phe Leu Al - #a Tyr Gln Asp Gln Ser     #  32605     - Phe Asp Ile Pro Asp Arg Thr Phe His Ser Th - #r Asn Thr Thr Trp Arg     #               32803270 - #                3275     - Leu Ser Ser Phe Glu Ser Met Thr Asp Val Ly - #s Glu Leu Ile Pro Glu     #              32950     - Phe Phe Tyr Leu Pro Glu Phe Leu Val Asn Ar - #g Glu Gly Phe Asp Phe     #          33105     - Gly Val Arg Gln Asn Gly Glu Arg Val Asn Hi - #s Val Asn Leu Pro Pro     #      33250     - Trp Ala Arg Asn Asp Pro Arg Leu Phe Ile Le - #u Ile His Arg Gln Ala     #  33405     - Leu Glu Ser Asp Tyr Val Ser Gln Asn Ile Cy - #s Gln Trp Ile Asp Leu     #               33603350 - #                3355     - Val Phe Gly Tyr Lys Gln Lys Gly Lys Ala Se - #r Val Gln Ala Ile Asn     #              33750     - Val Phe His Pro Ala Thr Tyr Phe Gly Met As - #p Val Ser Ala Val Glu     #          33905     - Asp Pro Val Gln Arg Arg Ala Leu Glu Thr Me - #t Ile Lys Thr Tyr Gly     #      34050     - Gln Thr Pro Arg Gln Leu Phe His Met Ala Hi - #s Val Ser Arg Pro Gly     #  34205     - Ala Lys Leu Asn Ile Glu Gly Glu Leu Pro Al - #a Ala Val Gly Leu Leu     #               34403430 - #                3435     - Val Gln Phe Ala Phe Arg Glu Thr Arg Glu Gl - #n Val Lys Glu Ile Thr     #              34550     - Tyr Pro Ser Pro Leu Ser Trp Ile Lys Gly Le - #u Lys Trp Gly Glu Tyr     #          34705     - Val Gly Ser Pro Ser Ala Pro Val Pro Val Va - #l Cys Phe Ser Gln Pro     #      34850     - His Gly Glu Arg Phe Gly Ser Leu Gln Ala Le - #u Pro Thr Arg Ala Ile     #  35005     - Cys Gly Leu Ser Arg Asn Phe Cys Leu Val Me - #t Thr Tyr Ser Lys Glu     #               35203510 - #                3515     - Gln Gly Val Arg Ser Met Asn Ser Thr Asp Il - #e Gln Trp Ser Ala Ile     #              35350     - Leu Ser Trp Gly Tyr Ala Asp Asn Ile Leu Ar - #g Leu Lys Ser Lys Gln     #          35505     - Ser Glu Pro Pro Val Asn Phe Ile Gln Ser Se - #r Gln Gln Tyr Gln Val     #      35650     - Thr Ser Cys Ala Trp Val Pro Asp Ser Cys Gl - #n Leu Phe Thr Gly Ser     #  35805     - Lys Cys Gly Val Ile Thr Ala Tyr Thr Asn Ar - #g Phe Thr Ser Ser Thr     #               36003590 - #                3595     - Pro Ser Glu Ile Glu Met Glu Thr Gln Ile Hi - #s Leu Tyr Gly His Thr     #              36150     - Glu Glu Ile Thr Ser Leu Phe Val Cys Lys Pr - #o Tyr Ser Ile Leu Ile     #          36305     - Ser Val Ser Arg Asp Gly Thr Cys Ile Ile Tr - #p Asp Leu Asn Arg Leu     #      36450     - Cys Tyr Val Gln Ser Leu Ala Gly His Lys Se - #r Pro Val Thr Ala Val     #  36605     - Ser Ala Ser Glu Thr Ser Gly Asp Ile Ala Th - #r Val Cys Asp Ser Ala     #               36803670 - #                3675     - Gly Gly Gly Ser Asp Leu Arg Leu Trp Thr Va - #l Asn Gly Asp Leu Val     #              36950     - Gly His Val His Cys Arg Glu Ile Ile Cys Se - #r Val Ala Phe Ser Asn     #          37105     - Gln Pro Glu Gly Val Ser Ile Asn Val Ile Al - #a Gly Gly Leu Glu Asn     #      37250     - Gly Ile Val Arg Leu Trp Ser Thr Trp Asp Le - #u Lys Pro Val Arg Glu     #  37405     - Ile Thr Phe Pro Lys Ser Asn Lys Pro Ile Il - #e Ser Leu Thr Phe Ser     #               37603750 - #                3755     - Cys Asp Gly His His Leu Tyr Thr Ala Asn Se - #r Asp Gly Thr Val Ile     #              37750     - Ala Trp Cys Arg Lys Asp Gln His Arg Leu Ly - #s Gln Pro Met Phe Tyr     #          37905     - Ser Phe Leu Ser Ser Tyr Ala Ala Gly     #       3800     - (2) INFORMATION FOR SEQ ID NO:11:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 12225 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (ix) FEATURE:               (A) NAME/KEY: CDS               (B) LOCATION: 190..11208     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:     - GCGGCCGCGT CGACGCGGCG GCGGCAGCGG CGTCGGCTCG GGGTTCTCCG GG - #AGAGGGGG       60     - AGTGCGCGGC GGCCGCAGCT GCCACAAACC AGGTGAAGCT TTGTTCTAAG AA - #TATTTGTT      120     - TCATCTAGTT TATGAGTCCA AATGATATAG ACTGTAAATG TCACAGCAGT GG - #TGAAAGAC      180     - TGCTCGGTC ATG AGC ACC GAC AGT AAC TCA CTG GCA - # CGT GAA TTT CTG      228     #Asn Ser Leu Ala Arg Glu Phe Leu     #         10     - ACC GAT GTC AAC CGG CTT TGC AAT GCA GTG GT - #C CAG AGG GTG GAG GCC      276     Thr Asp Val Asn Arg Leu Cys Asn Ala Val Va - #l Gln Arg Val Glu Ala     #     25     - AGG GAG GAA GAA GAG GAG GAG ACG CAC ATG GC - #A ACC CTT GGA CAG TAC      324     Arg Glu Glu Glu Glu Glu Glu Thr His Met Al - #a Thr Leu Gly Gln Tyr     # 45     - CTT GTC CAT GGT CGA GGA TTT CTA TTA CTT AC - #C AAG CTA AAT TCT ATA      372     Leu Val His Gly Arg Gly Phe Leu Leu Leu Th - #r Lys Leu Asn Ser Ile     #                 60     - ATT GAT CAG GCA TTG ACA TGT AGA GAA GAA CT - #C CTG ACT CTT CTT CTG      420     Ile Asp Gln Ala Leu Thr Cys Arg Glu Glu Le - #u Leu Thr Leu Leu Leu     #             75     - TCT CTC CTT CCA CTG GTA TGG AAG ATA CCT GT - #C CAA GAA GAA AAG GCA      468     Ser Leu Leu Pro Leu Val Trp Lys Ile Pro Va - #l Gln Glu Glu Lys Ala     #         90     - ACA GAT TTT AAC CTA CCG CTC TCA GCA GAT AT - #A ATC CTG ACC AAA GAA      516     Thr Asp Phe Asn Leu Pro Leu Ser Ala Asp Il - #e Ile Leu Thr Lys Glu     #    105     - AAG AAC TCA AGT TCA CAA AGA TCC ACT CAG GA - #A AAA TTA CAT TTA GAA      564     Lys Asn Ser Ser Ser Gln Arg Ser Thr Gln Gl - #u Lys Leu His Leu Glu     110                 1 - #15                 1 - #20                 1 -     #25     - GGA AGT GCC CTG TCT AGT CAG GTT TCT GCA AA - #A GTA AAT GTT TTT CGA      612     Gly Ser Ala Leu Ser Ser Gln Val Ser Ala Ly - #s Val Asn Val Phe Arg     #               140     - AAA AGC AGA CGA CAG CGT AAA ATT ACC CAT CG - #C TAT TCT GTA AGA GAT      660     Lys Ser Arg Arg Gln Arg Lys Ile Thr His Ar - #g Tyr Ser Val Arg Asp     #           155     - GCA AGA AAG ACA CAG CTC TCC ACC TCA GAT TC - #A GAA GCC AAT TCA GAT      708     Ala Arg Lys Thr Gln Leu Ser Thr Ser Asp Se - #r Glu Ala Asn Ser Asp     #       170     - GAA AAA GGC ATA GCA ATG AAT AAG CAT AGA AG - #G CCC CAT CTG CTG CAT      756     Glu Lys Gly Ile Ala Met Asn Lys His Arg Ar - #g Pro His Leu Leu His     #   185     - CAT TTT TTA ACA TCG TTT CCT AAA CAA GAC CA - #C CCC AAA GCT AAA CTT      804     His Phe Leu Thr Ser Phe Pro Lys Gln Asp Hi - #s Pro Lys Ala Lys Leu     190                 1 - #95                 2 - #00                 2 -     #05     - GAC CGC TTA GCA ACC AAA GAA CAG ACT CCT CC - #A GAT GCT ATG GCT TTG      852     Asp Arg Leu Ala Thr Lys Glu Gln Thr Pro Pr - #o Asp Ala Met Ala Leu     #               220     - GAA AAT TCC AGA GAG ATT ATT CCA AGA CAG GG - #G TCA AAC ACT GAC ATT      900     Glu Asn Ser Arg Glu Ile Ile Pro Arg Gln Gl - #y Ser Asn Thr Asp Ile     #           235     - TTA AGT GAG CCA GCT GCC TTG TCT GTT ATC AG - #T AAC ATG AAC AAT TCT      948     Leu Ser Glu Pro Ala Ala Leu Ser Val Ile Se - #r Asn Met Asn Asn Ser     #       250     - CCA TTT GAC TTA TGT CAT GTT TTG TTA TCT TT - #A TTA GAA AAA GTT TGT      996     Pro Phe Asp Leu Cys His Val Leu Leu Ser Le - #u Leu Glu Lys Val Cys     #   265     - AAG TTT GAC GTT ACC TTG AAT CAT AAT TCT CC - #T TTA GCA GCC AGT GTA     1044     Lys Phe Asp Val Thr Leu Asn His Asn Ser Pr - #o Leu Ala Ala Ser Val     270                 2 - #75                 2 - #80                 2 -     #85     - GTG CCC ACA CTA ACT GAA TTC CTA GCA GGC TT - #T GGG GAC TGC TGC AGT     1092     Val Pro Thr Leu Thr Glu Phe Leu Ala Gly Ph - #e Gly Asp Cys Cys Ser     #               300     - CTG AGC GAC AAC TTG GAG AGT CGA GTA GTT TC - #T GCA GGT TGG ACC GAA     1140     Leu Ser Asp Asn Leu Glu Ser Arg Val Val Se - #r Ala Gly Trp Thr Glu     #           315     - GAA CCG GTG GCT TTG ATT CAA AGG ATG CTC TT - #T CGA ACA GTG TTG CAT     1188     Glu Pro Val Ala Leu Ile Gln Arg Met Leu Ph - #e Arg Thr Val Leu His     #       330     - CTT CTG TCA GTA GAT GTT AGT ACT GCA GAG AT - #G ATG CCA GAA AAT CTT     1236     Leu Leu Ser Val Asp Val Ser Thr Ala Glu Me - #t Met Pro Glu Asn Leu     #   345     - AGG AAA AAT TTA ACT GAA TTG CTT AGA GCA GC - #T TTA AAA ATT AGA ATA     1284     Arg Lys Asn Leu Thr Glu Leu Leu Arg Ala Al - #a Leu Lys Ile Arg Ile     350                 3 - #55                 3 - #60                 3 -     #65     - TGC CTA GAA AAG CAG CCT GAC CCT TTT GCA CC - #A AGA CAA AAG AAA ACA     1332     Cys Leu Glu Lys Gln Pro Asp Pro Phe Ala Pr - #o Arg Gln Lys Lys Thr     #               380     - CTG CAG GAG GTT CAG GAA GAT TTT GTG TTT TC - #A AAG TAT CGT CAT AGA     1380     Leu Gln Glu Val Gln Glu Asp Phe Val Phe Se - #r Lys Tyr Arg His Arg     #           395     - GCC CTT CTT TTA CCT GAG CTT TTG GAA GGA GT - #T CTT CAG ATT CTG ATC     1428     Ala Leu Leu Leu Pro Glu Leu Leu Glu Gly Va - #l Leu Gln Ile Leu Ile     #       410     - TGT TGT CTT CAA AGT GCA GCT TCA AAT CCC TT - #C TAC TTC AGT CAA GCC     1476     Cys Cys Leu Gln Ser Ala Ala Ser Asn Pro Ph - #e Tyr Phe Ser Gln Ala     #   425     - ATG GAT TTG GTT CAA GAA TTC ATT CAG CAT CA - #T GGA TTT AAT TTA TTT     1524     Met Asp Leu Val Gln Glu Phe Ile Gln His Hi - #s Gly Phe Asn Leu Phe     430                 4 - #35                 4 - #40                 4 -     #45     - GAA ACA GCA GTT CTT CAA ATG GAA TGG CTG GT - #T TTA AGA GAT GGA GTT     1572     Glu Thr Ala Val Leu Gln Met Glu Trp Leu Va - #l Leu Arg Asp Gly Val     #               460     - CCT CCC GAG GCC TCA GAG CAT TTG AAA GCC CT - #A ATA AAT AGT GTG ATG     1620     Pro Pro Glu Ala Ser Glu His Leu Lys Ala Le - #u Ile Asn Ser Val Met     #           475     - AAA ATA ATG AGC ACT GTC AAA AAA GTG AAA TC - #A GAG CAA CTT CAT CAT     1668     Lys Ile Met Ser Thr Val Lys Lys Val Lys Se - #r Glu Gln Leu His His     #       490     - TCG ATG TGT ACA AGA AAA AGG CAC AGA CGA TG - #T GAA TAT TCT CAT TTT     1716     Ser Met Cys Thr Arg Lys Arg His Arg Arg Cy - #s Glu Tyr Ser His Phe     #   505     - ATG CAT CAT CAC CGA GAT CTC TCA GGT CTT CT - #G GTT TCG GCT TTT AAA     1764     Met His His His Arg Asp Leu Ser Gly Leu Le - #u Val Ser Ala Phe Lys     510                 5 - #15                 5 - #20                 5 -     #25     - AAC CAG GTT TCC AAA AAC CCA TTT GAA GAG AC - #T GCA GAT GGA GAT GTT     1812     Asn Gln Val Ser Lys Asn Pro Phe Glu Glu Th - #r Ala Asp Gly Asp Val     #               540     - TAT TAT CCT GAG CGG TGC TGT TGC ATT GCA GT - #G TGT GCC CAT CAG TGC     1860     Tyr Tyr Pro Glu Arg Cys Cys Cys Ile Ala Va - #l Cys Ala His Gln Cys     #           555     - TTG CGC TTA CTA CAG CAG GCT TCC TTG AGC AG - #C ACT TGT GTC CAG ATC     1908     Leu Arg Leu Leu Gln Gln Ala Ser Leu Ser Se - #r Thr Cys Val Gln Ile     #       570     - CTA TCG GGT GTT CAT AAC ATT GGA ATA TGC TG - #T TGT ATG GAT CCC AAA     1956     Leu Ser Gly Val His Asn Ile Gly Ile Cys Cy - #s Cys Met Asp Pro Lys     #   585     - TCT GTA ATC ATT CCT TTG CTC CAT GCT TTT AA - #A TTG CCA GCA CTG AAA     2004     Ser Val Ile Ile Pro Leu Leu His Ala Phe Ly - #s Leu Pro Ala Leu Lys     590                 5 - #95                 6 - #00                 6 -     #05     - AAT TTT CAG CAG CAT ATA TTG AAT ATC CTT AA - #C AAA CTT ATT TTG GAT     2052     Asn Phe Gln Gln His Ile Leu Asn Ile Leu As - #n Lys Leu Ile Leu Asp     #               620     - CAG TTA GGA GGA GCA GAG ATA TCA CCA AAA AT - #T AAA AAA GCA GCT TGT     2100     Gln Leu Gly Gly Ala Glu Ile Ser Pro Lys Il - #e Lys Lys Ala Ala Cys     #           635     - AAT ATT TGT ACT GTT GAC TCT GAC CAA CTA GC - #C CAA TTA GAA GAG ACA     2148     Asn Ile Cys Thr Val Asp Ser Asp Gln Leu Al - #a Gln Leu Glu Glu Thr     #       650     - CTG CAG GGA AAC TTA TGT GAT GCT GAA CTC TC - #C TCA AGT TTA TCC AGT     2196     Leu Gln Gly Asn Leu Cys Asp Ala Glu Leu Se - #r Ser Ser Leu Ser Ser     #   665     - CCT TCT TAC AGA TTT CAA GGG ATC CTG CCC AG - #C AGT GGA TCT GAA GAT     2244     Pro Ser Tyr Arg Phe Gln Gly Ile Leu Pro Se - #r Ser Gly Ser Glu Asp     670                 6 - #75                 6 - #80                 6 -     #85     - TTG TTG TGG AAA TGG GAT GCT TTA AAG GCT TA - #T CAG AAC TTT GTT TTT     2292     Leu Leu Trp Lys Trp Asp Ala Leu Lys Ala Ty - #r Gln Asn Phe Val Phe     #               700     - GAA GAA GAC AGA TTA CAT AGT ATA CAG ATT GC - #A AAT CAC ATT TGC AAT     2340     Glu Glu Asp Arg Leu His Ser Ile Gln Ile Al - #a Asn His Ile Cys Asn     #           715     - TTA ATC CAG AAA GGC AAT ATA GTT GTT CAG TG - #G AAA TTA TAT AAT TAC     2388     Leu Ile Gln Lys Gly Asn Ile Val Val Gln Tr - #p Lys Leu Tyr Asn Tyr     #       730     - ATA TTT AAT CCT GTG CTC CAA AGA GGA GTT GA - #A TTA GCA CAT CAT TGT     2436     Ile Phe Asn Pro Val Leu Gln Arg Gly Val Gl - #u Leu Ala His His Cys     #   745     - CAA CAC CTA AGC GTT ACT TCA GCT CAA AGT CA - #T GTA TGT AGC CAT CAT     2484     Gln His Leu Ser Val Thr Ser Ala Gln Ser Hi - #s Val Cys Ser His His     750                 7 - #55                 7 - #60                 7 -     #65     - AAC CAG TGC TTG CCT CAG GAC GTG CTT CAG AT - #T TAT GTA AAA ACT CTG     2532     Asn Gln Cys Leu Pro Gln Asp Val Leu Gln Il - #e Tyr Val Lys Thr Leu     #               780     - CCT ATC CTG CTT AAA TCC AGG GTA ATA AGA GA - #T TTG TTT TTG AGT TGT     2580     Pro Ile Leu Leu Lys Ser Arg Val Ile Arg As - #p Leu Phe Leu Ser Cys     #           795     - AAT GGA GTA AGT CAA ATA ATC GAA TTA AAT TG - #C TTA AAT GGT ATT CGA     2628     Asn Gly Val Ser Gln Ile Ile Glu Leu Asn Cy - #s Leu Asn Gly Ile Arg     #       810     - AGT CAT TCT CTA AAA GCA TTT GAA ACT CTG AT - #A ATC AGC CTA GGG GAG     2676     Ser His Ser Leu Lys Ala Phe Glu Thr Leu Il - #e Ile Ser Leu Gly Glu     #   825     - CAA CAG AAA GAT GCC TCA GTT CCA GAT ATT GA - #T GGG ATA GAC ATT GAA     2724     Gln Gln Lys Asp Ala Ser Val Pro Asp Ile As - #p Gly Ile Asp Ile Glu     830                 8 - #35                 8 - #40                 8 -     #45     - CAG AAG GAG TTG TCC TCT GTA CAT GTG GGT AC - #T TCT TTT CAT CAT CAG     2772     Gln Lys Glu Leu Ser Ser Val His Val Gly Th - #r Ser Phe His His Gln     #               860     - CAA GCT TAT TCA GAT TCT CCT CAG AGT CTC AG - #C AAA TTT TAT GCT GGC     2820     Gln Ala Tyr Ser Asp Ser Pro Gln Ser Leu Se - #r Lys Phe Tyr Ala Gly     #           875     - CTC AAA GAA GCT TAT CCA AAG AGA CGG AAG AC - #T GTT AAC CAA GAT GTT     2868     Leu Lys Glu Ala Tyr Pro Lys Arg Arg Lys Th - #r Val Asn Gln Asp Val     #       890     - CAT ATC AAC ACA ATA AAC CTA TTC CTC TGT GT - #G GCT TTT TTA TGC GTA     2916     His Ile Asn Thr Ile Asn Leu Phe Leu Cys Va - #l Ala Phe Leu Cys Val     #   905     - AGT AAA GAA GCA GAG TCT GAC AGG GAG TCG GC - #C AAT GAC TCA GAA GAT     2964     Ser Lys Glu Ala Glu Ser Asp Arg Glu Ser Al - #a Asn Asp Ser Glu Asp     910                 9 - #15                 9 - #20                 9 -     #25     - ACT TCT GGC TAT GAC AGC ACA GCC AGC GAG CC - #T TTA AGT CAT ATG CTG     3012     Thr Ser Gly Tyr Asp Ser Thr Ala Ser Glu Pr - #o Leu Ser His Met Leu     #               940     - CCA TGT ATA TCT CTC GAG AGC CTT GTC TTG CC - #T TCT CCT GAA CAT ATG     3060     Pro Cys Ile Ser Leu Glu Ser Leu Val Leu Pr - #o Ser Pro Glu His Met     #           955     - CAC CAA GCA GCA GAC ATT TGG TCT ATG TGT CG - #T TGG ATC TAC ATG TTG     3108     His Gln Ala Ala Asp Ile Trp Ser Met Cys Ar - #g Trp Ile Tyr Met Leu     #       970     - AGT TCA GTG TTC CAG AAA CAG TTT TAT AGG CT - #T GGT GGT TTC CGA GTA     3156     Ser Ser Val Phe Gln Lys Gln Phe Tyr Arg Le - #u Gly Gly Phe Arg Val     #   985     - TGC CAT AAG TTA ATA TTT ATG ATA ATA CAG AA - #A CTG TTC AGA AGT CAC     3204     Cys His Lys Leu Ile Phe Met Ile Ile Gln Ly - #s Leu Phe Arg Ser His     990                 9 - #95                 1 - #000                1005     - AAA GAG GAG CAA GGA AAA AAG GAG GGA GAT AC - #A AGT GTA AAT GAA AAC     3252     Lys Glu Glu Gln Gly Lys Lys Glu Gly Asp Th - #r Ser Val Asn Glu Asn     #              10205     - CAG GAT TTA AAC AGA ATT TCT CAA CCT AAG AG - #A ACT ATG AAG GAA GAT     3300     Gln Asp Leu Asn Arg Ile Ser Gln Pro Lys Ar - #g Thr Met Lys Glu Asp     #          10350     - TTA TTA TCT TTG GCT ATA AAA AGT GAC CCC AT - #A CCA TCA GAA CTA GGT     3348     Leu Leu Ser Leu Ala Ile Lys Ser Asp Pro Il - #e Pro Ser Glu Leu Gly     #      10505     - AGT CTA AAA AAG AGT GCT GAC AGT TTA GGT AA - #A TTA GAG TTA CAG CAT     3396     Ser Leu Lys Lys Ser Ala Asp Ser Leu Gly Ly - #s Leu Glu Leu Gln His     #  10650     - ATT TCT TCC ATA AAT GTG GAA GAA GTT TCA GC - #T ACT GAA GCC GCT CCC     3444     Ile Ser Ser Ile Asn Val Glu Glu Val Ser Al - #a Thr Glu Ala Ala Pro     #               10851075 - #                1080     - GAG GAA GCA AAG CTA TTT ACA AGT CAA GAA AG - #T GAG ACC TCA CTT CAA     3492     Glu Glu Ala Lys Leu Phe Thr Ser Gln Glu Se - #r Glu Thr Ser Leu Gln     #              11005     - AGT ATA CGA CTT TTG GAA GCC CTT CTG GCC AT - #T TGT CTT CAT GGT GCC     3540     Ser Ile Arg Leu Leu Glu Ala Leu Leu Ala Il - #e Cys Leu His Gly Ala     #          11150     - AGA ACT AGT CAA CAG AAG ATG GAA TTG GAG TT - #A CCT AAT CAG AAC TTG     3588     Arg Thr Ser Gln Gln Lys Met Glu Leu Glu Le - #u Pro Asn Gln Asn Leu     #      11305     - TCT GTG GAA AGT ATA TTA TTT GAA ATG AGG GA - #C CAT CTT TCC CAG TCA     3636     Ser Val Glu Ser Ile Leu Phe Glu Met Arg As - #p His Leu Ser Gln Ser     #  11450     - AAG GTG ATT GAA ACA CAA CTA GCA AAG CCG TT - #A TTT GAT GCC CTG CTT     3684     Lys Val Ile Glu Thr Gln Leu Ala Lys Pro Le - #u Phe Asp Ala Leu Leu     #               11651155 - #                1160     - CGA GTT GCC CTC GGG AAT TAT TCA GCA GAT TT - #T GAA CAT AAT GAT GCT     3732     Arg Val Ala Leu Gly Asn Tyr Ser Ala Asp Ph - #e Glu His Asn Asp Ala     #              11805     - ATG ACT GAG AAG AGT CAT CAA TCT GCA GAA GA - #A TTG TCA TCC CAG CCT     3780     Met Thr Glu Lys Ser His Gln Ser Ala Glu Gl - #u Leu Ser Ser Gln Pro     #          11950     - GGT GAT TTT TCA GAA GAA GCT GAG GAT TCT CA - #G TGT TGT AGT TTT AAA     3828     Gly Asp Phe Ser Glu Glu Ala Glu Asp Ser Gl - #n Cys Cys Ser Phe Lys     #      12105     - CTT TTA GTT GAA GAA GAA GGT TAC GAA GCA GA - #T AGT GAA AGC AAT CCT     3876     Leu Leu Val Glu Glu Glu Gly Tyr Glu Ala As - #p Ser Glu Ser Asn Pro     #  12250     - GAA GAT GGC GAA ACC CAG GAT GAT GGG GTA GA - #C TTA AAG TCT GAA ACA     3924     Glu Asp Gly Glu Thr Gln Asp Asp Gly Val As - #p Leu Lys Ser Glu Thr     #               12451235 - #                1240     - GAA GGT TTC AGT GCA TCA AGC AGT CCA AAT GA - #C TTA CTC GAA AAC CTC     3972     Glu Gly Phe Ser Ala Ser Ser Ser Pro Asn As - #p Leu Leu Glu Asn Leu     #              12605     - ACT CAA GGG GAA ATA ATT TAT CCT GAG ATT TG - #T ATG CTG GAA TTA AAT     4020     Thr Gln Gly Glu Ile Ile Tyr Pro Glu Ile Cy - #s Met Leu Glu Leu Asn     #          12750     - TTG CTT TCT GCT AGT AAA GCC AAA CTT GAT GT - #G CTT GCC CAT GTA TTT     4068     Leu Leu Ser Ala Ser Lys Ala Lys Leu Asp Va - #l Leu Ala His Val Phe     #      12905     - GAG AGT TTT TTG AAA ATT ATT AGG CAG AAA GA - #A AAG AAT GTT TTT CTG     4116     Glu Ser Phe Leu Lys Ile Ile Arg Gln Lys Gl - #u Lys Asn Val Phe Leu     #  13050     - CTC ATG CAA CAG GGA ACT GTG AAA AAT CTT TT - #A GGA GGG TTC TTG AGT     4164     Leu Met Gln Gln Gly Thr Val Lys Asn Leu Le - #u Gly Gly Phe Leu Ser     #               13251315 - #                1320     - ATT TTA ACA CAG GAT GAT TCT GAT TTT CAA GC - #A TGC CAG AGA GTA TTG     4212     Ile Leu Thr Gln Asp Asp Ser Asp Phe Gln Al - #a Cys Gln Arg Val Leu     #              13405     - GTG GAT CTT TTG GTA TCT TTG ATG AGT TCA AG - #A ACA TGT TCA GAA GAG     4260     Val Asp Leu Leu Val Ser Leu Met Ser Ser Ar - #g Thr Cys Ser Glu Glu     #          13550     - CTA ACC CTT CTT TTG AGA ATA TTT CTG GAG AA - #A TCT CCT TGT ACA AAA     4308     Leu Thr Leu Leu Leu Arg Ile Phe Leu Glu Ly - #s Ser Pro Cys Thr Lys     #      13705     - ATT CTT CTT CTG GGT ATT CTG AAA ATT ATT GA - #A AGT GAT ACT ACT ATG     4356     Ile Leu Leu Leu Gly Ile Leu Lys Ile Ile Gl - #u Ser Asp Thr Thr Met     #  13850     - AGC CCT TCA CAG TAT CTA ACC TTC CCT TTA CT - #G CAC GCT CCA AAT TTA     4404     Ser Pro Ser Gln Tyr Leu Thr Phe Pro Leu Le - #u His Ala Pro Asn Leu     #               14051395 - #                1400     - AGC AAC GGT GTT TCA TCA CAA AAG TAT CCT GG - #G ATT TTA AAC AGT AAG     4452     Ser Asn Gly Val Ser Ser Gln Lys Tyr Pro Gl - #y Ile Leu Asn Ser Lys     #              14205     - GCC ATG GGT TTA TTG AGA AGA GCA CGA GTT TC - #A CGG AGC AAG AAA GAG     4500     Ala Met Gly Leu Leu Arg Arg Ala Arg Val Se - #r Arg Ser Lys Lys Glu     #          14350     - GCT GAT AGA GAG AGT TTT CCC CAT CGG CTG CT - #T TCA TCT TGG CAC ATA     4548     Ala Asp Arg Glu Ser Phe Pro His Arg Leu Le - #u Ser Ser Trp His Ile     #      14505     - GCC CCA GTC CAC CTG CCG TTG CTG GGG CAA AA - #C TGC TGG CCA CAC CTA     4596     Ala Pro Val His Leu Pro Leu Leu Gly Gln As - #n Cys Trp Pro His Leu     #  14650     - TCA GAA GGT TTC AGT GTT TCC CTG TGG TTT AA - #T GTG GAG TGT ATC CAT     4644     Ser Glu Gly Phe Ser Val Ser Leu Trp Phe As - #n Val Glu Cys Ile His     #               14851475 - #                1480     - GAA GCT GAG AGT ACT ACA GAA AAA GGA AAG AA - #G ATA AAG AAA AGA AAC     4692     Glu Ala Glu Ser Thr Thr Glu Lys Gly Lys Ly - #s Ile Lys Lys Arg Asn     #              15005     - AAA TCA TTA ATT TTA CCA GAT AGC AGT TTT GA - #T GGT ACA GAG AGC GAC     4740     Lys Ser Leu Ile Leu Pro Asp Ser Ser Phe As - #p Gly Thr Glu Ser Asp     #          15150     - AGA CCA GAA GGT GCA GAG TAC ATA AAT CCT GG - #T GAA AGA CTC ATA GAA     4788     Arg Pro Glu Gly Ala Glu Tyr Ile Asn Pro Gl - #y Glu Arg Leu Ile Glu     #      15305     - GAA GGA TGT ATT CAT ATA ATT TCA CTG GGA TC - #C AAA GCG TTG ATG ATC     4836     Glu Gly Cys Ile His Ile Ile Ser Leu Gly Se - #r Lys Ala Leu Met Ile     #  15450     - CAA GTG TGG GCT GAT CCC CAC AAT GCC ACT CT - #T ATC TTT CGT GTG TGC     4884     Gln Val Trp Ala Asp Pro His Asn Ala Thr Le - #u Ile Phe Arg Val Cys     #               15651555 - #                1560     - ATG GAT TCA AAT GAT GAC ATG AAA GCT GTT TT - #A CTA GCA CAG GTT GAA     4932     Met Asp Ser Asn Asp Asp Met Lys Ala Val Le - #u Leu Ala Gln Val Glu     #              15805     - TCA CAG GAG AAT ATT TTC CTC CCA AGC AAA TG - #G CAA CAT TTA GTA CTC     4980     Ser Gln Glu Asn Ile Phe Leu Pro Ser Lys Tr - #p Gln His Leu Val Leu     #          15950     - ACC TAC TTA CAG CAG CCC CAA GGG AAA AGG AG - #G ATT CAT GGG AAA ATC     5028     Thr Tyr Leu Gln Gln Pro Gln Gly Lys Arg Ar - #g Ile His Gly Lys Ile     #      16105     - TCC ATA TGG GTC TCT GGA CAG AGG AAG CCT GA - #T GTT ACT TTG GAT TTT     5076     Ser Ile Trp Val Ser Gly Gln Arg Lys Pro As - #p Val Thr Leu Asp Phe     #  16250     - ATG CTT CCA AGA AAA ACA AGT TTG TCA TCT GA - #T AGC AAT AAA ACA TTT     5124     Met Leu Pro Arg Lys Thr Ser Leu Ser Ser As - #p Ser Asn Lys Thr Phe     #               16451635 - #                1640     - TGC ATG ATT GGC CAT TGT TTA TCA TCC CAA GA - #A GAG TTT TTG CAG TTG     5172     Cys Met Ile Gly His Cys Leu Ser Ser Gln Gl - #u Glu Phe Leu Gln Leu     #              16605     - GCT GGA AAA TGG GAC CTG GGA AAT TTG CTT CT - #C TTC AAC GGA GCT AAG     5220     Ala Gly Lys Trp Asp Leu Gly Asn Leu Leu Le - #u Phe Asn Gly Ala Lys     #          16750     - GTT GGT TCA CAA GAG GCC TTT TAT CTG TAT GC - #T TGT GGA CCC AAC CAT     5268     Val Gly Ser Gln Glu Ala Phe Tyr Leu Tyr Al - #a Cys Gly Pro Asn His     #      16905     - ACA TCT GTA ATG CCA TGT AAG TAT GGC AAG CC - #A GTC AAT GAC TAC TCC     5316     Thr Ser Val Met Pro Cys Lys Tyr Gly Lys Pr - #o Val Asn Asp Tyr Ser     #  17050     - AAA TAT ATT AAT AAA GAA ATT TTG CGA TGT GA - #A CAA ATC AGA GAA CTT     5364     Lys Tyr Ile Asn Lys Glu Ile Leu Arg Cys Gl - #u Gln Ile Arg Glu Leu     #               17251715 - #                1720     - TTT ATG ACC AAG AAA GAT GTG GAT ATT GGT CT - #C TTA ATT GAA AGT CTT     5412     Phe Met Thr Lys Lys Asp Val Asp Ile Gly Le - #u Leu Ile Glu Ser Leu     #              17405     - TCA GTT GTT TAT ACA ACT TAC TGT CCT GCT CA - #G TAT ACC ATC TAT GAA     5460     Ser Val Val Tyr Thr Thr Tyr Cys Pro Ala Gl - #n Tyr Thr Ile Tyr Glu     #          17550     - CCA GTG ATT AGA CTT AAA GGT CAA ATG AAA AC - #C CAA CTC TCT CAA AGA     5508     Pro Val Ile Arg Leu Lys Gly Gln Met Lys Th - #r Gln Leu Ser Gln Arg     #      17705     - CCC TTC AGC TCA AAA GAA GTT CAG AGC ATC TT - #A TTA GAA CCT CAT CAT     5556     Pro Phe Ser Ser Lys Glu Val Gln Ser Ile Le - #u Leu Glu Pro His His     #  17850     - CTA AAA AAT CTC CAA CCT ACT GAA TAT AAA AC - #T ATT CAA GGC ATT CTG     5604     Leu Lys Asn Leu Gln Pro Thr Glu Tyr Lys Th - #r Ile Gln Gly Ile Leu     #               18051795 - #                1800     - CAC GAA ATT GGT GGA ACT GGC ATA TTT GTT TT - #T CTC TTT GCC AGG GTT     5652     His Glu Ile Gly Gly Thr Gly Ile Phe Val Ph - #e Leu Phe Ala Arg Val     #              18205     - GTT GAA CTC AGT AGC TGT GAA GAA ACT CAA GC - #A TTA GCA CTG CGA GTT     5700     Val Glu Leu Ser Ser Cys Glu Glu Thr Gln Al - #a Leu Ala Leu Arg Val     #          18350     - ATA CTC TCA TTA ATT AAA TAC AAC CAA CAA AG - #A GTA CAT GAA TTA GAA     5748     Ile Leu Ser Leu Ile Lys Tyr Asn Gln Gln Ar - #g Val His Glu Leu Glu     #      18505     - AAT TGT AAT GGA CTT TCT ATG ATT CAT CAG GT - #G TTG ATC AAA CAA AAA     5796     Asn Cys Asn Gly Leu Ser Met Ile His Gln Va - #l Leu Ile Lys Gln Lys     #  18650     - TGC ATT GTT GGG TTT TAC ATT TTG AAG ACC CT - #T CTT GAA GGA TGC TGT     5844     Cys Ile Val Gly Phe Tyr Ile Leu Lys Thr Le - #u Leu Glu Gly Cys Cys     #               18851875 - #                1880     - GGT GAA GAT ATT ATT TAT ATG AAT GAG AAT GG - #A GAG TTT AAG TTG GAT     5892     Gly Glu Asp Ile Ile Tyr Met Asn Glu Asn Gl - #y Glu Phe Lys Leu Asp     #              19005     - GTA GAC TCT AAT GCT ATA ATC CAA GAT GTT AA - #G CTG TTA GAG GAA CTA     5940     Val Asp Ser Asn Ala Ile Ile Gln Asp Val Ly - #s Leu Leu Glu Glu Leu     #          19150     - TTG CTT GAC TGG AAG ATA TGG AGT AAA GCA GA - #G CAA GGT GTT TGG GAA     5988     Leu Leu Asp Trp Lys Ile Trp Ser Lys Ala Gl - #u Gln Gly Val Trp Glu     #      19305     - ACT TTG CTA GCA GCT CTA GAA GTC CTC ATC AG - #A GCA GAT CAC CAC CAG     6036     Thr Leu Leu Ala Ala Leu Glu Val Leu Ile Ar - #g Ala Asp His His Gln     #  19450     - CAG ATG TTT AAT ATT AAG CAG TTA TTG AAA GC - #T CAA GTG GTT CAT CAC     6084     Gln Met Phe Asn Ile Lys Gln Leu Leu Lys Al - #a Gln Val Val His His     #               19651955 - #                1960     - TTT CTA CTG ACT TGT CAG GTT TTG CAG GAA TA - #C AAA GAG GGG CAA CTC     6132     Phe Leu Leu Thr Cys Gln Val Leu Gln Glu Ty - #r Lys Glu Gly Gln Leu     #              19805     - ACA CCC ATG CCC CGA GAG GTT TGT AGA TCA TT - #T GTG AAA ATT ATA GCA     6180     Thr Pro Met Pro Arg Glu Val Cys Arg Ser Ph - #e Val Lys Ile Ile Ala     #          19950     - GAA GTC CTT GGA TCT CCT CCA GAT TTG GAA TT - #A TTG ACA ATT ATC TTC     6228     Glu Val Leu Gly Ser Pro Pro Asp Leu Glu Le - #u Leu Thr Ile Ile Phe     #      20105     - AAT TTC CTT TTA GCA GTT CAC CCT CCT ACT AA - #T ACT TAC GTT TGT CAC     6276     Asn Phe Leu Leu Ala Val His Pro Pro Thr As - #n Thr Tyr Val Cys His     #  20250     - AAT CCC ACG AAC TTC TAC TTT TCT TTG CAC AT - #A GAT GGC AAG ATC TTT     6324     Asn Pro Thr Asn Phe Tyr Phe Ser Leu His Il - #e Asp Gly Lys Ile Phe     #               20452035 - #                2040     - CAG GAG AAA GTG CGG TCA ATC ATG TAC CTG AG - #G CAT TCC AGC AGT GGA     6372     Gln Glu Lys Val Arg Ser Ile Met Tyr Leu Ar - #g His Ser Ser Ser Gly     #              20605     - GGA AGG TCC CTT ATG AGC CCT GGA TTT ATG GT - #A ATA AGC CCA TCT GGT     6420     Gly Arg Ser Leu Met Ser Pro Gly Phe Met Va - #l Ile Ser Pro Ser Gly     #          20750     - TTT ACT GCT TCA CCA TAT GAA GGA GAG AAT TC - #C TCT AAT ATT ATT CCA     6468     Phe Thr Ala Ser Pro Tyr Glu Gly Glu Asn Se - #r Ser Asn Ile Ile Pro     #      20905     - CAA CAG ATG GCC GCC CAT ATG CTG CGT TCT AG - #A AGC CTA CCA GCA TTC     6516     Gln Gln Met Ala Ala His Met Leu Arg Ser Ar - #g Ser Leu Pro Ala Phe     #  21050     - CCT ACT TCT TCA CTA CTA ACG CAA TCA CAA AA - #A CTG ACT GGA AGT TTG     6564     Pro Thr Ser Ser Leu Leu Thr Gln Ser Gln Ly - #s Leu Thr Gly Ser Leu     #               21252115 - #                2120     - GGT TGT AGT ATC GAC AGG TTA CAA AAT ATT GC - #A GAT ACT TAT GTT GCC     6612     Gly Cys Ser Ile Asp Arg Leu Gln Asn Ile Al - #a Asp Thr Tyr Val Ala     #              21405     - ACC CAA TCA AAG AAA CAA AAT TCT TTG GGG AG - #T TCC GAC ACA CTG AAA     6660     Thr Gln Ser Lys Lys Gln Asn Ser Leu Gly Se - #r Ser Asp Thr Leu Lys     #          21550     - AAA GGC AAA GAG GAC GCA TTC ATC AGT AGC TG - #T GAG TCT GCA AAA ACT     6708     Lys Gly Lys Glu Asp Ala Phe Ile Ser Ser Cy - #s Glu Ser Ala Lys Thr     #      21705     - GTT TGT GAA ATG GAA GCT GTC CTC TCA GCC CA - #G GTC TCT GTC AGT GAT     6756     Val Cys Glu Met Glu Ala Val Leu Ser Ala Gl - #n Val Ser Val Ser Asp     #  21850     - GTC CCA AAG GGA GTG CTG GGA TTT CCA GTG GT - #C AAA GCA GAT CAT AAA     6804     Val Pro Lys Gly Val Leu Gly Phe Pro Val Va - #l Lys Ala Asp His Lys     #               22052195 - #                2200     - CAG TTG GGA GCA GAA CCC AGG TCA GAA GAT GA - #C AGT CCT GGG GAT GAG     6852     Gln Leu Gly Ala Glu Pro Arg Ser Glu Asp As - #p Ser Pro Gly Asp Glu     #              22205     - TCC TGC CCA CGC CGA CCT GAT TAC CTA AAG GG - #A TTG GCC TCC TTC CAG     6900     Ser Cys Pro Arg Arg Pro Asp Tyr Leu Lys Gl - #y Leu Ala Ser Phe Gln     #          22350     - CGA AGC CAC AGC ACT ATT GCA AGC CTT GGG CT - #A GCT TTT CCT TCA CAG     6948     Arg Ser His Ser Thr Ile Ala Ser Leu Gly Le - #u Ala Phe Pro Ser Gln     #      22505     - AAC GGA TCT GCA GCT GTT GGC CGT TGG CCA AG - #T CTT GTT GAT AGA AAC     6996     Asn Gly Ser Ala Ala Val Gly Arg Trp Pro Se - #r Leu Val Asp Arg Asn     #  22650     - ACT GAT GAT TGG GAA AAC TTT GCC TAT TCT CT - #T GGT TAT GAG CCA AAT     7044     Thr Asp Asp Trp Glu Asn Phe Ala Tyr Ser Le - #u Gly Tyr Glu Pro Asn     #               22852275 - #                2280     - TAC AAC CGA ACT GCA AGT GCT CAC AGT GTA AC - #T GAA GAC TGT TTG GTA     7092     Tyr Asn Arg Thr Ala Ser Ala His Ser Val Th - #r Glu Asp Cys Leu Val     #              23005     - CCT ATA TGC TGT GGA TTA TAT GAA CTC CTA AG - #T GGG GTT CTT CTT ATC     7140     Pro Ile Cys Cys Gly Leu Tyr Glu Leu Leu Se - #r Gly Val Leu Leu Ile     #          23150     - CTG CCT GAT GTT TTG CTT GAA GAT GTG ATG GA - #C AAG CTT ATT CAA GCA     7188     Leu Pro Asp Val Leu Leu Glu Asp Val Met As - #p Lys Leu Ile Gln Ala     #      23305     - GAT ACA CTT TTG GTC CTC GTT AAC CAC CCA TC - #A CCA GCT ATA CAA CAA     7236     Asp Thr Leu Leu Val Leu Val Asn His Pro Se - #r Pro Ala Ile Gln Gln     #  23450     - GGT GTT ATT AAA CTA TTA GAT GCA TAT TTT GC - #T AGA GCA TCT AAG GAA     7284     Gly Val Ile Lys Leu Leu Asp Ala Tyr Phe Al - #a Arg Ala Ser Lys Glu     #               23652355 - #                2360     - CAA AAA GAT AAA TTT CTG AAG AAT CGT GGA TT - #T TCC TTG CTA GCC AAC     7332     Gln Lys Asp Lys Phe Leu Lys Asn Arg Gly Ph - #e Ser Leu Leu Ala Asn     #              23805     - CAG TTG TAT CTT CAT CGA GGA ACT CAA GAA TT - #G TTA GAA TGC TTC ATC     7380     Gln Leu Tyr Leu His Arg Gly Thr Gln Glu Le - #u Leu Glu Cys Phe Ile     #          23950     - GAA ATG TTC TTT GGT CGA CAT ATT GGC CTT GA - #T GAA GAA TTT GAT CTG     7428     Glu Met Phe Phe Gly Arg His Ile Gly Leu As - #p Glu Glu Phe Asp Leu     #      24105     - GAA GAT GTG AGA AAC ATG GGA TTG TTT CAG AA - #G TGG TCT GTC ATT CCT     7476     Glu Asp Val Arg Asn Met Gly Leu Phe Gln Ly - #s Trp Ser Val Ile Pro     #  24250     - ATT CTG GGA CTA ATA GAG ACC TCT CTA TAT GA - #C AAC ATA CTC TTG CAT     7524     Ile Leu Gly Leu Ile Glu Thr Ser Leu Tyr As - #p Asn Ile Leu Leu His     #               24452435 - #                2440     - AAT GCT CTT TTA CTT CTT CCC CAT CAT GCA GT - #A GTT CAA AAG CGG AAA     7572     Asn Ala Leu Leu Leu Leu Pro His His Ala Va - #l Val Gln Lys Arg Lys     #              24605     - AGC ATT GCT GGT CCT CGA AAA TTT CCC CTT GC - #T CAA ACT GAA TCG CTT     7620     Ser Ile Ala Gly Pro Arg Lys Phe Pro Leu Al - #a Gln Thr Glu Ser Leu     #          24750     - CTG ATG AAA ATG CGT TCA GTG GCA AAT GAT GA - #G CTT CAT GTG ATG ATG     7668     Leu Met Lys Met Arg Ser Val Ala Asn Asp Gl - #u Leu His Val Met Met     #      24905     - CAA CGG AGA ATG AGC CAA GAG AAC CCT AGC CA - #A GCA ACT GAA ACG GAA     7716     Gln Arg Arg Met Ser Gln Glu Asn Pro Ser Gl - #n Ala Thr Glu Thr Glu     #  25050     - CTT GCG CAG AGA CTA CAG AGG CTC ACT GTT TT - #A GCA GTC AAC AGG ATT     7764     Leu Ala Gln Arg Leu Gln Arg Leu Thr Val Le - #u Ala Val Asn Arg Ile     #               25252515 - #                2520     - ATT TAT CAA GAA TTT AAT TCA GAC ATT ATT GA - #C ATT TTG AGA ACT CCA     7812     Ile Tyr Gln Glu Phe Asn Ser Asp Ile Ile As - #p Ile Leu Arg Thr Pro     #              25405     - GAA AAT GTA ACT CAA AGC AAG ACC TCA GTT TT - #C CAG ACC GAA ATT TCT     7860     Glu Asn Val Thr Gln Ser Lys Thr Ser Val Ph - #e Gln Thr Glu Ile Ser     #          25550     - GAG GAA AAT ATT CAT CAT GAA CAG TCT TCT GT - #T TTC AAT CCA TTT CAG     7908     Glu Glu Asn Ile His His Glu Gln Ser Ser Va - #l Phe Asn Pro Phe Gln     #      25705     - AAA GAA ATT TTT ACA TAT CTG GTA GAA GGA TT - #C AAA GTA TCT ATT GGT     7956     Lys Glu Ile Phe Thr Tyr Leu Val Glu Gly Ph - #e Lys Val Ser Ile Gly     #  25850     - TCA AGT AAA GCC AGT GGT TCC AAG CAG CAA TG - #G ACT AAA ATT CTG TGG     8004     Ser Ser Lys Ala Ser Gly Ser Lys Gln Gln Tr - #p Thr Lys Ile Leu Trp     #               26052595 - #                2600     - TCT TGT AAG GAG ACC TTC CGA ATG CAG CTT GG - #G AGA CTA CTA GTG CAT     8052     Ser Cys Lys Glu Thr Phe Arg Met Gln Leu Gl - #y Arg Leu Leu Val His     #              26205     - ATT TTG TCG CCA GCC CAC GCT GCA CAA GAG AG - #A AAG CAA ATT TTT GAA     8100     Ile Leu Ser Pro Ala His Ala Ala Gln Glu Ar - #g Lys Gln Ile Phe Glu     #          26350     - ATA GTT CAT GAA CCA AAT CAT CAG GAA ATA CT - #A CGA GAC TGT CTC AGC     8148     Ile Val His Glu Pro Asn His Gln Glu Ile Le - #u Arg Asp Cys Leu Ser     #      26505     - CCA TCC CTA CAA CAT GGA GCC AAG TTA GTT TT - #G TAT TTG TCA GAG TTG     8196     Pro Ser Leu Gln His Gly Ala Lys Leu Val Le - #u Tyr Leu Ser Glu Leu     #  26650     - ATA CAT AAT CAC CAA GGT GAA TTG ACT GAA GA - #A GAG CTA GGC ACA GCA     8244     Ile His Asn His Gln Gly Glu Leu Thr Glu Gl - #u Glu Leu Gly Thr Ala     #               26852675 - #                2680     - GAA CTG CTT ATG AAT GCT TTG AAG TTA TGT GG - #T CAC AAG TGC ATC CCT     8292     Glu Leu Leu Met Asn Ala Leu Lys Leu Cys Gl - #y His Lys Cys Ile Pro     #              27005     - CCC AGT GCA TCA ACA AAA GCA GAC CTT ATT AA - #A ATG ATC AAA GAG GAA     8340     Pro Ser Ala Ser Thr Lys Ala Asp Leu Ile Ly - #s Met Ile Lys Glu Glu     #          27150     - CAA AAG AAA TAT GAA ACT GAA GAA GGA GTG AA - #T AAA GCT GCT TGG CAG     8388     Gln Lys Lys Tyr Glu Thr Glu Glu Gly Val As - #n Lys Ala Ala Trp Gln     #      27305     - AAA ACA GTT AAC AAT AAT CAA CAA AGT CTC TT - #T CAG CGT CTG GAT TCA     8436     Lys Thr Val Asn Asn Asn Gln Gln Ser Leu Ph - #e Gln Arg Leu Asp Ser     #  27450     - AAA TCA AAG GAT ATA TCT AAA ATA GCT GCA GA - #T ATC ACC CAG GCA GTG     8484     Lys Ser Lys Asp Ile Ser Lys Ile Ala Ala As - #p Ile Thr Gln Ala Val     #               27652755 - #                2760     - TCT CTC TCC CAA GGA AAT GAG AGA AAA AAG GT - #G ATC CAG CAT ATT AGA     8532     Ser Leu Ser Gln Gly Asn Glu Arg Lys Lys Va - #l Ile Gln His Ile Arg     #              27805     - GGA ATG TAT AAA GTA GAT TTG AGT GCC AGC AG - #A CAT TGG CAG GAA CTT     8580     Gly Met Tyr Lys Val Asp Leu Ser Ala Ser Ar - #g His Trp Gln Glu Leu     #          27950     - ATT CAG CAG CTG ACA CAT GAT AGA GCA GTA TG - #G TAT GAC CCC ATC TAC     8628     Ile Gln Gln Leu Thr His Asp Arg Ala Val Tr - #p Tyr Asp Pro Ile Tyr     #      28105     - TAT CCA ACC TCA TGG CAG TTG GAT CCA ACA GA - #A GGG CCA AAT CGA GAG     8676     Tyr Pro Thr Ser Trp Gln Leu Asp Pro Thr Gl - #u Gly Pro Asn Arg Glu     #  28250     - AGG AGA CGT TTA CAG AGA TGT TAT TTA ACT AT - #T CCA AAT AAG TAT CTC     8724     Arg Arg Arg Leu Gln Arg Cys Tyr Leu Thr Il - #e Pro Asn Lys Tyr Leu     #               28452835 - #                2840     - CTT AGG GAT AGA CAG AAA TCA GAA GAT GTT GT - #C AAA CCA CCA CTC TCT     8772     Leu Arg Asp Arg Gln Lys Ser Glu Asp Val Va - #l Lys Pro Pro Leu Ser     #              28605     - TAC CTG TTT GAA GAC AAA ACT CAT TCT TCT TT - #C TCT TCT ACT GTC AAA     8820     Tyr Leu Phe Glu Asp Lys Thr His Ser Ser Ph - #e Ser Ser Thr Val Lys     #          28750     - GAC AAA GCT GCA AGT GAA TCT ATA AGA GTG AA - #T CGA AGA TGC ATC AGT     8868     Asp Lys Ala Ala Ser Glu Ser Ile Arg Val As - #n Arg Arg Cys Ile Ser     #      28905     - GTT GCA CCA TCT AGA GAG ACA GCT GGT GAA TT - #G TTA CTA GGT AAA TGT     8916     Val Ala Pro Ser Arg Glu Thr Ala Gly Glu Le - #u Leu Leu Gly Lys Cys     #  29050     - GGA ATG TAT TTT GTG GAA GAT AAT GCT TCT GA - #T ACA GTT GAA AGT TCG     8964     Gly Met Tyr Phe Val Glu Asp Asn Ala Ser As - #p Thr Val Glu Ser Ser     #               29252915 - #                2920     - AGC CTT CAG GGA GAG TTG GAA CCA GCA TCA TT - #T TCC TGG ACA TAT GAA     9012     Ser Leu Gln Gly Glu Leu Glu Pro Ala Ser Ph - #e Ser Trp Thr Tyr Glu     #              29405     - GAA ATT AAA GAA GTT CAC AAG CGT TGG TGG CA - #A TTG AGA GAT AAT GCT     9060     Glu Ile Lys Glu Val His Lys Arg Trp Trp Gl - #n Leu Arg Asp Asn Ala     #          29550     - GTA GAA ATC TTT CTA ACA AAT GGC AGA ACA CT - #C CTG TTG GCA TTT GAT     9108     Val Glu Ile Phe Leu Thr Asn Gly Arg Thr Le - #u Leu Leu Ala Phe Asp     #      29705     - AAC ACC AAG GTT CGT GAT GAT GTA TAC CAC AA - #T ATA CTC ACA AAT AAC     9156     Asn Thr Lys Val Arg Asp Asp Val Tyr His As - #n Ile Leu Thr Asn Asn     #  29850     - CTC CCT AAT CTT CTG GAA TAT GGT AAC ATC AC - #C GCT CTG ACA AAT TTA     9204     Leu Pro Asn Leu Leu Glu Tyr Gly Asn Ile Th - #r Ala Leu Thr Asn Leu     #               30052995 - #                3000     - TGG TAT ACT GGG CAA ATT ACT AAT TTT GAA TA - #T TTG ACT CAC TTA AAC     9252     Trp Tyr Thr Gly Gln Ile Thr Asn Phe Glu Ty - #r Leu Thr His Leu Asn     #              30205     - AAA CAT GCT GGC CGA TCC TTC AAT GAT CTC AT - #G CAG TAT CCT GTG TTC     9300     Lys His Ala Gly Arg Ser Phe Asn Asp Leu Me - #t Gln Tyr Pro Val Phe     #          30350     - CCA TTT ATA CTT GCT GAC TAC GTT AGT GAG AC - #A CTT GAC CTC AAT GAT     9348     Pro Phe Ile Leu Ala Asp Tyr Val Ser Glu Th - #r Leu Asp Leu Asn Asp     #      30505     - CTG TTG ATA TAC AGA AAT CTC TCT AAA CCT AT - #A GCT GTT CAG TAT AAA     9396     Leu Leu Ile Tyr Arg Asn Leu Ser Lys Pro Il - #e Ala Val Gln Tyr Lys     #  30650     - GAA AAA GAA GAT CGT TAT GTG GAC ACA TAC AA - #G TAC TTG GAG GAA GAG     9444     Glu Lys Glu Asp Arg Tyr Val Asp Thr Tyr Ly - #s Tyr Leu Glu Glu Glu     #               30853075 - #                3080     - TAC CGC AAA GGA GCC AGA GAA GAT GAC CCC AT - #G CCT CCC GTG CAG CCC     9492     Tyr Arg Lys Gly Ala Arg Glu Asp Asp Pro Me - #t Pro Pro Val Gln Pro     #              31005     - TAT CAC TAT GGC TCC CAC TAT TCC AAT AGC GG - #C ACT GTG CTT CAC TTC     9540     Tyr His Tyr Gly Ser His Tyr Ser Asn Ser Gl - #y Thr Val Leu His Phe     #          31150     - CTG GTC AGG ATG CCT CCT TTC ACT AAA ATG TT - #T TTA GCC TAT CAA GAT     9588     Leu Val Arg Met Pro Pro Phe Thr Lys Met Ph - #e Leu Ala Tyr Gln Asp     #      31305     - CAA AGT TTT GAC ATT CCA GAC AGA ACT TTT CA - #T TCT ACA AAT ACA ACT     9636     Gln Ser Phe Asp Ile Pro Asp Arg Thr Phe Hi - #s Ser Thr Asn Thr Thr     #  31450     - TGG CGA CTC TCA TCT TTT GAA TCT ATG ACT GA - #T GTG AAA GAA CTT ATC     9684     Trp Arg Leu Ser Ser Phe Glu Ser Met Thr As - #p Val Lys Glu Leu Ile     #               31653155 - #                3160     - CCA GAG TTT TTC TAT CTT CCA GAG TTC CTA GT - #T AAC CGT GAA GGT TTT     9732     Pro Glu Phe Phe Tyr Leu Pro Glu Phe Leu Va - #l Asn Arg Glu Gly Phe     #              31805     - GAT TTT GGT GTG CGT CAG AAT GGT GAA CGG GT - #T AAT CAC GTC AAC CTT     9780     Asp Phe Gly Val Arg Gln Asn Gly Glu Arg Va - #l Asn His Val Asn Leu     #          31950     - CCC CCT TGG GCG CGT AAT GAT CCT CGT CTT TT - #T ATC CTC ATC CAT CGG     9828     Pro Pro Trp Ala Arg Asn Asp Pro Arg Leu Ph - #e Ile Leu Ile His Arg     #      32105     - CAG GCT CTA GAG TCT GAC TAC GTG TCG CAG AA - #C ATC TGT CAG TGG ATT     9876     Gln Ala Leu Glu Ser Asp Tyr Val Ser Gln As - #n Ile Cys Gln Trp Ile     #  32250     - GAC TTG GTG TTT GGG TAT AAG CAA AAG GGG AA - #G GCT TCT GTT CAA GCG     9924     Asp Leu Val Phe Gly Tyr Lys Gln Lys Gly Ly - #s Ala Ser Val Gln Ala     #               32453235 - #                3240     - ATC AAT GTT TTT CAT CCT GCT ACA TAT TTT GG - #A ATG GAT GTC TCT GCA     9972     Ile Asn Val Phe His Pro Ala Thr Tyr Phe Gl - #y Met Asp Val Ser Ala     #              32605     - GTT GAA GAT CCA GTT CAG AGA CGA GCG CTA GA - #A ACC ATG ATA AAA ACC     10020     Val Glu Asp Pro Val Gln Arg Arg Ala Leu Gl - #u Thr Met Ile Lys Thr     #          32750     - TAC GGG CAG ACT CCC CGT CAG CTG TTC CAC AT - #G GCC CAT GTG AGC AGA     10068     Tyr Gly Gln Thr Pro Arg Gln Leu Phe His Me - #t Ala His Val Ser Arg     #      32905     - CCT GGA GCC AAG CTC AAT ATT GAA GGA GAG CT - #T CCA GCT GCT GTG GGG     10116     Pro Gly Ala Lys Leu Asn Ile Glu Gly Glu Le - #u Pro Ala Ala Val Gly     #  33050     - TTG CTA GTG CAG TTT GCT TTC AGG GAG ACC CG - #A GAA CAG GTC AAA GAA     10164     Leu Leu Val Gln Phe Ala Phe Arg Glu Thr Ar - #g Glu Gln Val Lys Glu     #               33253315 - #                3320     - ATC ACC TAT CCG AGT CCT TTG TCA TGG ATA AA - #A GGC TTG AAA TGG GGG     10212     Ile Thr Tyr Pro Ser Pro Leu Ser Trp Ile Ly - #s Gly Leu Lys Trp Gly     #              33405     - GAA TAC GTG GGT TCC CCC AGT GCT CCA GTA CC - #T GTG GTC TGC TTC AGC     10260     Glu Tyr Val Gly Ser Pro Ser Ala Pro Val Pr - #o Val Val Cys Phe Ser     #          33550     - CAG CCC CAC GGA GAA AGA TTT GGC TCT CTC CA - #G GCT CTG CCC ACC AGA     10308     Gln Pro His Gly Glu Arg Phe Gly Ser Leu Gl - #n Ala Leu Pro Thr Arg     #      33705     - GCA ATC TGT GGT TTG TCA CGG AAT TTC TGT CT - #T GTG ATG ACA TAT AGC     10356     Ala Ile Cys Gly Leu Ser Arg Asn Phe Cys Le - #u Val Met Thr Tyr Ser     #  33850     - AAG GAA CAA GGT GTG AGA AGC ATG AAC AGT AC - #G GAC ATT CAG TGG TCA     10404     Lys Glu Gln Gly Val Arg Ser Met Asn Ser Th - #r Asp Ile Gln Trp Ser     #               34053395 - #                3400     - GCC ATC CTG AGC TGG GGA TAT GCT GAT AAT AT - #T TTA AGG TTG AAG AGT     10452     Ala Ile Leu Ser Trp Gly Tyr Ala Asp Asn Il - #e Leu Arg Leu Lys Ser     #              34205     - AAA CAA AGT GAG CCT CCA GTA AAC TTT ATT CA - #A AGT TCA CAA CAG TAC     10500     Lys Gln Ser Glu Pro Pro Val Asn Phe Ile Gl - #n Ser Ser Gln Gln Tyr     #          34350     - CAG GTG ACT AGT TGT GCT TGG GTG CCT GAC AG - #T TGC CAG CTG TTT ACT     10548     Gln Val Thr Ser Cys Ala Trp Val Pro Asp Se - #r Cys Gln Leu Phe Thr     #      34505     - GGA AGC AAA TGC GGT GTC ATC ACA GCC TAC AC - #A AAC AGA TTT ACA AGC     10596     Gly Ser Lys Cys Gly Val Ile Thr Ala Tyr Th - #r Asn Arg Phe Thr Ser     #  34650     - AGC ACG CCA TCA GAA ATA GAA ATG GAG ACT CA - #A ATA CAT CTC TAT GGT     10644     Ser Thr Pro Ser Glu Ile Glu Met Glu Thr Gl - #n Ile His Leu Tyr Gly     #               34853475 - #                3480     - CAC ACA GAA GAG ATA ACC AGC TTA TTT GTT TG - #C AAA CCA TAC AGT ATA     10692     His Thr Glu Glu Ile Thr Ser Leu Phe Val Cy - #s Lys Pro Tyr Ser Ile     #              35005     - CTG ATA AGT GTG AGC AGA GAC GGA ACC TGC AT - #C ATA TGG GAT TTA AAC     10740     Leu Ile Ser Val Ser Arg Asp Gly Thr Cys Il - #e Ile Trp Asp Leu Asn     #          35150     - AGG TTA TGC TAT GTA CAA AGT CTG GCG GGA CA - #C AAA AGC CCT GTC ACA     10788     Arg Leu Cys Tyr Val Gln Ser Leu Ala Gly Hi - #s Lys Ser Pro Val Thr     #      35305     - GCT GTC TCT GCC AGT GAA ACC TCA GGT GAT AT - #T GCT ACT GTG TGT GAT     10836     Ala Val Ser Ala Ser Glu Thr Ser Gly Asp Il - #e Ala Thr Val Cys Asp     #  35450     - TCA GCT GGC GGA GGC AGT GAC CTC AGA CTC TG - #G ACG GTG AAC GGG GAT     10884     Ser Ala Gly Gly Gly Ser Asp Leu Arg Leu Tr - #p Thr Val Asn Gly Asp     #               35653555 - #                3560     - CTC GTT GGA CAT GTC CAC TGC AGG GAG ATC AT - #C TGT TCC GTG GCT TTC     10932     Leu Val Gly His Val His Cys Arg Glu Ile Il - #e Cys Ser Val Ala Phe     #              35805     - TCC AAC CAG CCT GAG GGA GTA TCT ATC AAT GT - #A ATC GCT GGG GGA TTA     10980     Ser Asn Gln Pro Glu Gly Val Ser Ile Asn Va - #l Ile Ala Gly Gly Leu     #          35950     - GAA AAT GGA ATT GTT AGG TTA TGG AGC ACA TG - #G GAC TTA AAG CCT GTG     11028     Glu Asn Gly Ile Val Arg Leu Trp Ser Thr Tr - #p Asp Leu Lys Pro Val     #      36105     - AGA GAA ATT ACA TTT CCC AAA TCA AAT AAG CC - #C ATC ATC AGC CTT ACA     11076     Arg Glu Ile Thr Phe Pro Lys Ser Asn Lys Pr - #o Ile Ile Ser Leu Thr     #  36250     - TTT TCT TGT GAT GGC CAC CAT TTG TAC ACA GC - #A AAC AGT GAT GGG ACC     11124     Phe Ser Cys Asp Gly His His Leu Tyr Thr Al - #a Asn Ser Asp Gly Thr     #               36453635 - #                3640     - GTG ATT GCC TGG TGT CGG AAG GAC CAG CAC CG - #C TTG AAA CAG CCA ATG     11172     Val Ile Ala Trp Cys Arg Lys Asp Gln His Ar - #g Leu Lys Gln Pro Met     #              36605     - TTC TAT TCC TTC CTT AGC AGC TAT GCA GCC GG - #G TGA ATGCGAATGA     11218     Phe Tyr Ser Phe Leu Ser Ser Tyr Ala Ala Gl - #y  *     #           3670     - ACTTCATGTT CTCCAAAGCA CTTTAACTCC AAACTAGATT TGTTGACTTC AC - #CAGTTTTA     11278     - GGAGGTTGAA CCTAAAGAAA TGGATGACTG GACAAACCAT CCAAATAATG AT - #AAAGTCTA     11338     - TTCATCTGCA CAAAATTCTG AAGAGTCACA TGATCCTAAG AGGAAAGTTC TG - #TTCTATTT     11398     - TAGTGATAAT CTGGAAGATT GTGTCAATAT GCACTAGCCA ACAAGTTTTA AG - #CCTCGCAT     11458     - GGTACATTAA AATGATATTC TTAAAATTTT TTCCCACCAA GGTATTCCAA AG - #AAAATATT     11518     - AAGGTCTCCC CTTTTTCTAT GATTCCAAAA GGACCAGTAG AATTTAAATT GG - #TTGGTTGA     11578     - TGTTTATATA AAACACACTA AAATTATATT TTAAAAGTTT ATGCCTGAAA TA - #CTCCTCCC     11638     - ACCACACACA CATGCTCCAA AAGAGGAAAG AAAAAAAGAT AATTTTTAGG AC - #TTGATAAT     11698     - TGCTTTCTTT GAGAAGCAAA TTATTCAGTA GGTGCCTCTG TACCAAATAT TT - #TATGGAAT     11758     - ATCTAAATAC TAAAATAAAC TATGAATGAA TCTCAAAATT AGGCAGTTTT TG - #CCAGTTGC     11818     - TTTCTTAGCT CAAAGGAGAA CCAGAATTTT TTTGACAGCC ACAAACAAGA AT - #ACAGGTAT     11878     - CTTGGATTTC AGACACATTC TGTTTCTTCA TAAAAATTTT ACTTAAAATC TG - #TAACGCTA     11938     - GATATTGACT ATCCTTAGTT GAGTCACTGA GGTTTAAACA CAATGGTAAG TC - #TTAAAGTC     11998     - TGCTATTTAC AGAGCATTGA ATCTGTACCA ATTTGCAATA GAAAGCCTTC AG - #TATGCAAG     12058     - AAGTTTGCAT GGGTATTAAG AACACAGCCT AAATAAGGCA TTTGATTAAT CT - #GCAGGAAG     12118     - AATTTTCTTC CCCAAAACAG AATTATAAAA GCTTACTTTA AACAGGAGGC AG - #AATAATTC     12178     #             12225CATT CTGTTTCTAC TAACCTATAC CATCTGA     - (2) INFORMATION FOR SEQ ID NO:12:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 3672 amino               (B) TYPE: amino acid               (D) TOPOLOGY: unknown     -     (ii) MOLECULE TYPE: protein     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:     - Met Ser Thr Asp Ser Asn Ser Leu Ala Arg Gl - #u Phe Leu Thr Asp Val     #                 15     - Asn Arg Leu Cys Asn Ala Val Val Gln Arg Va - #l Glu Ala Arg Glu Glu     #             30     - Glu Glu Glu Glu Thr His Met Ala Thr Leu Gl - #y Gln Tyr Leu Val His     #         45     - Gly Arg Gly Phe Leu Leu Leu Thr Lys Leu As - #n Ser Ile Ile Asp Gln     #     60     - Ala Leu Thr Cys Arg Glu Glu Leu Leu Thr Le - #u Leu Leu Ser Leu Leu     # 80     - Pro Leu Val Trp Lys Ile Pro Val Gln Glu Gl - #u Lys Ala Thr Asp Phe     #                 95     - Asn Leu Pro Leu Ser Ala Asp Ile Ile Leu Th - #r Lys Glu Lys Asn Ser     #           110     - Ser Ser Gln Arg Ser Thr Gln Glu Lys Leu Hi - #s Leu Glu Gly Ser Ala     #       125     - Leu Ser Ser Gln Val Ser Ala Lys Val Asn Va - #l Phe Arg Lys Ser Arg     #   140     - Arg Gln Arg Lys Ile Thr His Arg Tyr Ser Va - #l Arg Asp Ala Arg Lys     145                 1 - #50                 1 - #55                 1 -     #60     - Thr Gln Leu Ser Thr Ser Asp Ser Glu Ala As - #n Ser Asp Glu Lys Gly     #               175     - Ile Ala Met Asn Lys His Arg Arg Pro His Le - #u Leu His His Phe Leu     #           190     - Thr Ser Phe Pro Lys Gln Asp His Pro Lys Al - #a Lys Leu Asp Arg Leu     #       205     - Ala Thr Lys Glu Gln Thr Pro Pro Asp Ala Me - #t Ala Leu Glu Asn Ser     #   220     - Arg Glu Ile Ile Pro Arg Gln Gly Ser Asn Th - #r Asp Ile Leu Ser Glu     225                 2 - #30                 2 - #35                 2 -     #40     - Pro Ala Ala Leu Ser Val Ile Ser Asn Met As - #n Asn Ser Pro Phe Asp     #               255     - Leu Cys His Val Leu Leu Ser Leu Leu Glu Ly - #s Val Cys Lys Phe Asp     #           270     - Val Thr Leu Asn His Asn Ser Pro Leu Ala Al - #a Ser Val Val Pro Thr     #       285     - Leu Thr Glu Phe Leu Ala Gly Phe Gly Asp Cy - #s Cys Ser Leu Ser Asp     #   300     - Asn Leu Glu Ser Arg Val Val Ser Ala Gly Tr - #p Thr Glu Glu Pro Val     305                 3 - #10                 3 - #15                 3 -     #20     - Ala Leu Ile Gln Arg Met Leu Phe Arg Thr Va - #l Leu His Leu Leu Ser     #               335     - Val Asp Val Ser Thr Ala Glu Met Met Pro Gl - #u Asn Leu Arg Lys Asn     #           350     - Leu Thr Glu Leu Leu Arg Ala Ala Leu Lys Il - #e Arg Ile Cys Leu Glu     #       365     - Lys Gln Pro Asp Pro Phe Ala Pro Arg Gln Ly - #s Lys Thr Leu Gln Glu     #   380     - Val Gln Glu Asp Phe Val Phe Ser Lys Tyr Ar - #g His Arg Ala Leu Leu     385                 3 - #90                 3 - #95                 4 -     #00     - Leu Pro Glu Leu Leu Glu Gly Val Leu Gln Il - #e Leu Ile Cys Cys Leu     #               415     - Gln Ser Ala Ala Ser Asn Pro Phe Tyr Phe Se - #r Gln Ala Met Asp Leu     #           430     - Val Gln Glu Phe Ile Gln His His Gly Phe As - #n Leu Phe Glu Thr Ala     #       445     - Val Leu Gln Met Glu Trp Leu Val Leu Arg As - #p Gly Val Pro Pro Glu     #   460     - Ala Ser Glu His Leu Lys Ala Leu Ile Asn Se - #r Val Met Lys Ile Met     465                 4 - #70                 4 - #75                 4 -     #80     - Ser Thr Val Lys Lys Val Lys Ser Glu Gln Le - #u His His Ser Met Cys     #               495     - Thr Arg Lys Arg His Arg Arg Cys Glu Tyr Se - #r His Phe Met His His     #           510     - His Arg Asp Leu Ser Gly Leu Leu Val Ser Al - #a Phe Lys Asn Gln Val     #       525     - Ser Lys Asn Pro Phe Glu Glu Thr Ala Asp Gl - #y Asp Val Tyr Tyr Pro     #   540     - Glu Arg Cys Cys Cys Ile Ala Val Cys Ala Hi - #s Gln Cys Leu Arg Leu     545                 5 - #50                 5 - #55                 5 -     #60     - Leu Gln Gln Ala Ser Leu Ser Ser Thr Cys Va - #l Gln Ile Leu Ser Gly     #               575     - Val His Asn Ile Gly Ile Cys Cys Cys Met As - #p Pro Lys Ser Val Ile     #           590     - Ile Pro Leu Leu His Ala Phe Lys Leu Pro Al - #a Leu Lys Asn Phe Gln     #       605     - Gln His Ile Leu Asn Ile Leu Asn Lys Leu Il - #e Leu Asp Gln Leu Gly     #   620     - Gly Ala Glu Ile Ser Pro Lys Ile Lys Lys Al - #a Ala Cys Asn Ile Cys     625                 6 - #30                 6 - #35                 6 -     #40     - Thr Val Asp Ser Asp Gln Leu Ala Gln Leu Gl - #u Glu Thr Leu Gln Gly     #               655     - Asn Leu Cys Asp Ala Glu Leu Ser Ser Ser Le - #u Ser Ser Pro Ser Tyr     #           670     - Arg Phe Gln Gly Ile Leu Pro Ser Ser Gly Se - #r Glu Asp Leu Leu Trp     #       685     - Lys Trp Asp Ala Leu Lys Ala Tyr Gln Asn Ph - #e Val Phe Glu Glu Asp     #   700     - Arg Leu His Ser Ile Gln Ile Ala Asn His Il - #e Cys Asn Leu Ile Gln     705                 7 - #10                 7 - #15                 7 -     #20     - Lys Gly Asn Ile Val Val Gln Trp Lys Leu Ty - #r Asn Tyr Ile Phe Asn     #               735     - Pro Val Leu Gln Arg Gly Val Glu Leu Ala Hi - #s His Cys Gln His Leu     #           750     - Ser Val Thr Ser Ala Gln Ser His Val Cys Se - #r His His Asn Gln Cys     #       765     - Leu Pro Gln Asp Val Leu Gln Ile Tyr Val Ly - #s Thr Leu Pro Ile Leu     #   780     - Leu Lys Ser Arg Val Ile Arg Asp Leu Phe Le - #u Ser Cys Asn Gly Val     785                 7 - #90                 7 - #95                 8 -     #00     - Ser Gln Ile Ile Glu Leu Asn Cys Leu Asn Gl - #y Ile Arg Ser His Ser     #               815     - Leu Lys Ala Phe Glu Thr Leu Ile Ile Ser Le - #u Gly Glu Gln Gln Lys     #           830     - Asp Ala Ser Val Pro Asp Ile Asp Gly Ile As - #p Ile Glu Gln Lys Glu     #       845     - Leu Ser Ser Val His Val Gly Thr Ser Phe Hi - #s His Gln Gln Ala Tyr     #   860     - Ser Asp Ser Pro Gln Ser Leu Ser Lys Phe Ty - #r Ala Gly Leu Lys Glu     865                 8 - #70                 8 - #75                 8 -     #80     - Ala Tyr Pro Lys Arg Arg Lys Thr Val Asn Gl - #n Asp Val His Ile Asn     #               895     - Thr Ile Asn Leu Phe Leu Cys Val Ala Phe Le - #u Cys Val Ser Lys Glu     #           910     - Ala Glu Ser Asp Arg Glu Ser Ala Asn Asp Se - #r Glu Asp Thr Ser Gly     #       925     - Tyr Asp Ser Thr Ala Ser Glu Pro Leu Ser Hi - #s Met Leu Pro Cys Ile     #   940     - Ser Leu Glu Ser Leu Val Leu Pro Ser Pro Gl - #u His Met His Gln Ala     945                 9 - #50                 9 - #55                 9 -     #60     - Ala Asp Ile Trp Ser Met Cys Arg Trp Ile Ty - #r Met Leu Ser Ser Val     #               975     - Phe Gln Lys Gln Phe Tyr Arg Leu Gly Gly Ph - #e Arg Val Cys His Lys     #           990     - Leu Ile Phe Met Ile Ile Gln Lys Leu Phe Ar - #g Ser His Lys Glu Glu     #      10050     - Gln Gly Lys Lys Glu Gly Asp Thr Ser Val As - #n Glu Asn Gln Asp Leu     #  10205     - Asn Arg Ile Ser Gln Pro Lys Arg Thr Met Ly - #s Glu Asp Leu Leu Ser     #               10401030 - #                1035     - Leu Ala Ile Lys Ser Asp Pro Ile Pro Ser Gl - #u Leu Gly Ser Leu Lys     #              10550     - Lys Ser Ala Asp Ser Leu Gly Lys Leu Glu Le - #u Gln His Ile Ser Ser     #          10705     - Ile Asn Val Glu Glu Val Ser Ala Thr Glu Al - #a Ala Pro Glu Glu Ala     #      10850     - Lys Leu Phe Thr Ser Gln Glu Ser Glu Thr Se - #r Leu Gln Ser Ile Arg     #  11005     - Leu Leu Glu Ala Leu Leu Ala Ile Cys Leu Hi - #s Gly Ala Arg Thr Ser     #               11201110 - #                1115     - Gln Gln Lys Met Glu Leu Glu Leu Pro Asn Gl - #n Asn Leu Ser Val Glu     #              11350     - Ser Ile Leu Phe Glu Met Arg Asp His Leu Se - #r Gln Ser Lys Val Ile     #          11505     - Glu Thr Gln Leu Ala Lys Pro Leu Phe Asp Al - #a Leu Leu Arg Val Ala     #      11650     - Leu Gly Asn Tyr Ser Ala Asp Phe Glu His As - #n Asp Ala Met Thr Glu     #  11805     - Lys Ser His Gln Ser Ala Glu Glu Leu Ser Se - #r Gln Pro Gly Asp Phe     #               12001190 - #                1195     - Ser Glu Glu Ala Glu Asp Ser Gln Cys Cys Se - #r Phe Lys Leu Leu Val     #              12150     - Glu Glu Glu Gly Tyr Glu Ala Asp Ser Glu Se - #r Asn Pro Glu Asp Gly     #          12305     - Glu Thr Gln Asp Asp Gly Val Asp Leu Lys Se - #r Glu Thr Glu Gly Phe     #      12450     - Ser Ala Ser Ser Ser Pro Asn Asp Leu Leu Gl - #u Asn Leu Thr Gln Gly     #  12605     - Glu Ile Ile Tyr Pro Glu Ile Cys Met Leu Gl - #u Leu Asn Leu Leu Ser     #               12801270 - #                1275     - Ala Ser Lys Ala Lys Leu Asp Val Leu Ala Hi - #s Val Phe Glu Ser Phe     #              12950     - Leu Lys Ile Ile Arg Gln Lys Glu Lys Asn Va - #l Phe Leu Leu Met Gln     #          13105     - Gln Gly Thr Val Lys Asn Leu Leu Gly Gly Ph - #e Leu Ser Ile Leu Thr     #      13250     - Gln Asp Asp Ser Asp Phe Gln Ala Cys Gln Ar - #g Val Leu Val Asp Leu     #  13405     - Leu Val Ser Leu Met Ser Ser Arg Thr Cys Se - #r Glu Glu Leu Thr Leu     #               13601350 - #                1355     - Leu Leu Arg Ile Phe Leu Glu Lys Ser Pro Cy - #s Thr Lys Ile Leu Leu     #              13750     - Leu Gly Ile Leu Lys Ile Ile Glu Ser Asp Th - #r Thr Met Ser Pro Ser     #          13905     - Gln Tyr Leu Thr Phe Pro Leu Leu His Ala Pr - #o Asn Leu Ser Asn Gly     #      14050     - Val Ser Ser Gln Lys Tyr Pro Gly Ile Leu As - #n Ser Lys Ala Met Gly     #  14205     - Leu Leu Arg Arg Ala Arg Val Ser Arg Ser Ly - #s Lys Glu Ala Asp Arg     #               14401430 - #                1435     - Glu Ser Phe Pro His Arg Leu Leu Ser Ser Tr - #p His Ile Ala Pro Val     #              14550     - His Leu Pro Leu Leu Gly Gln Asn Cys Trp Pr - #o His Leu Ser Glu Gly     #          14705     - Phe Ser Val Ser Leu Trp Phe Asn Val Glu Cy - #s Ile His Glu Ala Glu     #      14850     - Ser Thr Thr Glu Lys Gly Lys Lys Ile Lys Ly - #s Arg Asn Lys Ser Leu     #  15005     - Ile Leu Pro Asp Ser Ser Phe Asp Gly Thr Gl - #u Ser Asp Arg Pro Glu     #               15201510 - #                1515     - Gly Ala Glu Tyr Ile Asn Pro Gly Glu Arg Le - #u Ile Glu Glu Gly Cys     #              15350     - Ile His Ile Ile Ser Leu Gly Ser Lys Ala Le - #u Met Ile Gln Val Trp     #          15505     - Ala Asp Pro His Asn Ala Thr Leu Ile Phe Ar - #g Val Cys Met Asp Ser     #      15650     - Asn Asp Asp Met Lys Ala Val Leu Leu Ala Gl - #n Val Glu Ser Gln Glu     #  15805     - Asn Ile Phe Leu Pro Ser Lys Trp Gln His Le - #u Val Leu Thr Tyr Leu     #               16001590 - #                1595     - Gln Gln Pro Gln Gly Lys Arg Arg Ile His Gl - #y Lys Ile Ser Ile Trp     #              16150     - Val Ser Gly Gln Arg Lys Pro Asp Val Thr Le - #u Asp Phe Met Leu Pro     #          16305     - Arg Lys Thr Ser Leu Ser Ser Asp Ser Asn Ly - #s Thr Phe Cys Met Ile     #      16450     - Gly His Cys Leu Ser Ser Gln Glu Glu Phe Le - #u Gln Leu Ala Gly Lys     #  16605     - Trp Asp Leu Gly Asn Leu Leu Leu Phe Asn Gl - #y Ala Lys Val Gly Ser     #               16801670 - #                1675     - Gln Glu Ala Phe Tyr Leu Tyr Ala Cys Gly Pr - #o Asn His Thr Ser Val     #              16950     - Met Pro Cys Lys Tyr Gly Lys Pro Val Asn As - #p Tyr Ser Lys Tyr Ile     #          17105     - Asn Lys Glu Ile Leu Arg Cys Glu Gln Ile Ar - #g Glu Leu Phe Met Thr     #      17250     - Lys Lys Asp Val Asp Ile Gly Leu Leu Ile Gl - #u Ser Leu Ser Val Val     #  17405     - Tyr Thr Thr Tyr Cys Pro Ala Gln Tyr Thr Il - #e Tyr Glu Pro Val Ile     #               17601750 - #                1755     - Arg Leu Lys Gly Gln Met Lys Thr Gln Leu Se - #r Gln Arg Pro Phe Ser     #              17750     - Ser Lys Glu Val Gln Ser Ile Leu Leu Glu Pr - #o His His Leu Lys Asn     #          17905     - Leu Gln Pro Thr Glu Tyr Lys Thr Ile Gln Gl - #y Ile Leu His Glu Ile     #      18050     - Gly Gly Thr Gly Ile Phe Val Phe Leu Phe Al - #a Arg Val Val Glu Leu     #  18205     - Ser Ser Cys Glu Glu Thr Gln Ala Leu Ala Le - #u Arg Val Ile Leu Ser     #               18401830 - #                1835     - Leu Ile Lys Tyr Asn Gln Gln Arg Val His Gl - #u Leu Glu Asn Cys Asn     #              18550     - Gly Leu Ser Met Ile His Gln Val Leu Ile Ly - #s Gln Lys Cys Ile Val     #          18705     - Gly Phe Tyr Ile Leu Lys Thr Leu Leu Glu Gl - #y Cys Cys Gly Glu Asp     #      18850     - Ile Ile Tyr Met Asn Glu Asn Gly Glu Phe Ly - #s Leu Asp Val Asp Ser     #  19005     - Asn Ala Ile Ile Gln Asp Val Lys Leu Leu Gl - #u Glu Leu Leu Leu Asp     #               19201910 - #                1915     - Trp Lys Ile Trp Ser Lys Ala Glu Gln Gly Va - #l Trp Glu Thr Leu Leu     #              19350     - Ala Ala Leu Glu Val Leu Ile Arg Ala Asp Hi - #s His Gln Gln Met Phe     #          19505     - Asn Ile Lys Gln Leu Leu Lys Ala Gln Val Va - #l His His Phe Leu Leu     #      19650     - Thr Cys Gln Val Leu Gln Glu Tyr Lys Glu Gl - #y Gln Leu Thr Pro Met     #  19805     - Pro Arg Glu Val Cys Arg Ser Phe Val Lys Il - #e Ile Ala Glu Val Leu     #               20001990 - #                1995     - Gly Ser Pro Pro Asp Leu Glu Leu Leu Thr Il - #e Ile Phe Asn Phe Leu     #              20150     - Leu Ala Val His Pro Pro Thr Asn Thr Tyr Va - #l Cys His Asn Pro Thr     #          20305     - Asn Phe Tyr Phe Ser Leu His Ile Asp Gly Ly - #s Ile Phe Gln Glu Lys     #      20450     - Val Arg Ser Ile Met Tyr Leu Arg His Ser Se - #r Ser Gly Gly Arg Ser     #  20605     - Leu Met Ser Pro Gly Phe Met Val Ile Ser Pr - #o Ser Gly Phe Thr Ala     #               20802070 - #                2075     - Ser Pro Tyr Glu Gly Glu Asn Ser Ser Asn Il - #e Ile Pro Gln Gln Met     #              20950     - Ala Ala His Met Leu Arg Ser Arg Ser Leu Pr - #o Ala Phe Pro Thr Ser     #          21105     - Ser Leu Leu Thr Gln Ser Gln Lys Leu Thr Gl - #y Ser Leu Gly Cys Ser     #      21250     - Ile Asp Arg Leu Gln Asn Ile Ala Asp Thr Ty - #r Val Ala Thr Gln Ser     #  21405     - Lys Lys Gln Asn Ser Leu Gly Ser Ser Asp Th - #r Leu Lys Lys Gly Lys     #               21602150 - #                2155     - Glu Asp Ala Phe Ile Ser Ser Cys Glu Ser Al - #a Lys Thr Val Cys Glu     #              21750     - Met Glu Ala Val Leu Ser Ala Gln Val Ser Va - #l Ser Asp Val Pro Lys     #          21905     - Gly Val Leu Gly Phe Pro Val Val Lys Ala As - #p His Lys Gln Leu Gly     #      22050     - Ala Glu Pro Arg Ser Glu Asp Asp Ser Pro Gl - #y Asp Glu Ser Cys Pro     #  22205     - Arg Arg Pro Asp Tyr Leu Lys Gly Leu Ala Se - #r Phe Gln Arg Ser His     #               22402230 - #                2235     - Ser Thr Ile Ala Ser Leu Gly Leu Ala Phe Pr - #o Ser Gln Asn Gly Ser     #              22550     - Ala Ala Val Gly Arg Trp Pro Ser Leu Val As - #p Arg Asn Thr Asp Asp     #          22705     - Trp Glu Asn Phe Ala Tyr Ser Leu Gly Tyr Gl - #u Pro Asn Tyr Asn Arg     #      22850     - Thr Ala Ser Ala His Ser Val Thr Glu Asp Cy - #s Leu Val Pro Ile Cys     #  23005     - Cys Gly Leu Tyr Glu Leu Leu Ser Gly Val Le - #u Leu Ile Leu Pro Asp     #               23202310 - #                2315     - Val Leu Leu Glu Asp Val Met Asp Lys Leu Il - #e Gln Ala Asp Thr Leu     #              23350     - Leu Val Leu Val Asn His Pro Ser Pro Ala Il - #e Gln Gln Gly Val Ile     #          23505     - Lys Leu Leu Asp Ala Tyr Phe Ala Arg Ala Se - #r Lys Glu Gln Lys Asp     #      23650     - Lys Phe Leu Lys Asn Arg Gly Phe Ser Leu Le - #u Ala Asn Gln Leu Tyr     #  23805     - Leu His Arg Gly Thr Gln Glu Leu Leu Glu Cy - #s Phe Ile Glu Met Phe     #               24002390 - #                2395     - Phe Gly Arg His Ile Gly Leu Asp Glu Glu Ph - #e Asp Leu Glu Asp Val     #              24150     - Arg Asn Met Gly Leu Phe Gln Lys Trp Ser Va - #l Ile Pro Ile Leu Gly     #          24305     - Leu Ile Glu Thr Ser Leu Tyr Asp Asn Ile Le - #u Leu His Asn Ala Leu     #      24450     - Leu Leu Leu Pro His His Ala Val Val Gln Ly - #s Arg Lys Ser Ile Ala     #  24605     - Gly Pro Arg Lys Phe Pro Leu Ala Gln Thr Gl - #u Ser Leu Leu Met Lys     #               24802470 - #                2475     - Met Arg Ser Val Ala Asn Asp Glu Leu His Va - #l Met Met Gln Arg Arg     #              24950     - Met Ser Gln Glu Asn Pro Ser Gln Ala Thr Gl - #u Thr Glu Leu Ala Gln     #          25105     - Arg Leu Gln Arg Leu Thr Val Leu Ala Val As - #n Arg Ile Ile Tyr Gln     #      25250     - Glu Phe Asn Ser Asp Ile Ile Asp Ile Leu Ar - #g Thr Pro Glu Asn Val     #  25405     - Thr Gln Ser Lys Thr Ser Val Phe Gln Thr Gl - #u Ile Ser Glu Glu Asn     #               25602550 - #                2555     - Ile His His Glu Gln Ser Ser Val Phe Asn Pr - #o Phe Gln Lys Glu Ile     #              25750     - Phe Thr Tyr Leu Val Glu Gly Phe Lys Val Se - #r Ile Gly Ser Ser Lys     #          25905     - Ala Ser Gly Ser Lys Gln Gln Trp Thr Lys Il - #e Leu Trp Ser Cys Lys     #      26050     - Glu Thr Phe Arg Met Gln Leu Gly Arg Leu Le - #u Val His Ile Leu Ser     #  26205     - Pro Ala His Ala Ala Gln Glu Arg Lys Gln Il - #e Phe Glu Ile Val His     #               26402630 - #                2635     - Glu Pro Asn His Gln Glu Ile Leu Arg Asp Cy - #s Leu Ser Pro Ser Leu     #              26550     - Gln His Gly Ala Lys Leu Val Leu Tyr Leu Se - #r Glu Leu Ile His Asn     #          26705     - His Gln Gly Glu Leu Thr Glu Glu Glu Leu Gl - #y Thr Ala Glu Leu Leu     #      26850     - Met Asn Ala Leu Lys Leu Cys Gly His Lys Cy - #s Ile Pro Pro Ser Ala     #  27005     - Ser Thr Lys Ala Asp Leu Ile Lys Met Ile Ly - #s Glu Glu Gln Lys Lys     #               27202710 - #                2715     - Tyr Glu Thr Glu Glu Gly Val Asn Lys Ala Al - #a Trp Gln Lys Thr Val     #              27350     - Asn Asn Asn Gln Gln Ser Leu Phe Gln Arg Le - #u Asp Ser Lys Ser Lys     #          27505     - Asp Ile Ser Lys Ile Ala Ala Asp Ile Thr Gl - #n Ala Val Ser Leu Ser     #      27650     - Gln Gly Asn Glu Arg Lys Lys Val Ile Gln Hi - #s Ile Arg Gly Met Tyr     #  27805     - Lys Val Asp Leu Ser Ala Ser Arg His Trp Gl - #n Glu Leu Ile Gln Gln     #               28002790 - #                2795     - Leu Thr His Asp Arg Ala Val Trp Tyr Asp Pr - #o Ile Tyr Tyr Pro Thr     #              28150     - Ser Trp Gln Leu Asp Pro Thr Glu Gly Pro As - #n Arg Glu Arg Arg Arg     #          28305     - Leu Gln Arg Cys Tyr Leu Thr Ile Pro Asn Ly - #s Tyr Leu Leu Arg Asp     #      28450     - Arg Gln Lys Ser Glu Asp Val Val Lys Pro Pr - #o Leu Ser Tyr Leu Phe     #  28605     - Glu Asp Lys Thr His Ser Ser Phe Ser Ser Th - #r Val Lys Asp Lys Ala     #               28802870 - #                2875     - Ala Ser Glu Ser Ile Arg Val Asn Arg Arg Cy - #s Ile Ser Val Ala Pro     #              28950     - Ser Arg Glu Thr Ala Gly Glu Leu Leu Leu Gl - #y Lys Cys Gly Met Tyr     #          29105     - Phe Val Glu Asp Asn Ala Ser Asp Thr Val Gl - #u Ser Ser Ser Leu Gln     #      29250     - Gly Glu Leu Glu Pro Ala Ser Phe Ser Trp Th - #r Tyr Glu Glu Ile Lys     #  29405     - Glu Val His Lys Arg Trp Trp Gln Leu Arg As - #p Asn Ala Val Glu Ile     #               29602950 - #                2955     - Phe Leu Thr Asn Gly Arg Thr Leu Leu Leu Al - #a Phe Asp Asn Thr Lys     #              29750     - Val Arg Asp Asp Val Tyr His Asn Ile Leu Th - #r Asn Asn Leu Pro Asn     #          29905     - Leu Leu Glu Tyr Gly Asn Ile Thr Ala Leu Th - #r Asn Leu Trp Tyr Thr     #      30050     - Gly Gln Ile Thr Asn Phe Glu Tyr Leu Thr Hi - #s Leu Asn Lys His Ala     #  30205     - Gly Arg Ser Phe Asn Asp Leu Met Gln Tyr Pr - #o Val Phe Pro Phe Ile     #               30403030 - #                3035     - Leu Ala Asp Tyr Val Ser Glu Thr Leu Asp Le - #u Asn Asp Leu Leu Ile     #              30550     - Tyr Arg Asn Leu Ser Lys Pro Ile Ala Val Gl - #n Tyr Lys Glu Lys Glu     #          30705     - Asp Arg Tyr Val Asp Thr Tyr Lys Tyr Leu Gl - #u Glu Glu Tyr Arg Lys     #      30850     - Gly Ala Arg Glu Asp Asp Pro Met Pro Pro Va - #l Gln Pro Tyr His Tyr     #  31005     - Gly Ser His Tyr Ser Asn Ser Gly Thr Val Le - #u His Phe Leu Val Arg     #               31203110 - #                3115     - Met Pro Pro Phe Thr Lys Met Phe Leu Ala Ty - #r Gln Asp Gln Ser Phe     #              31350     - Asp Ile Pro Asp Arg Thr Phe His Ser Thr As - #n Thr Thr Trp Arg Leu     #          31505     - Ser Ser Phe Glu Ser Met Thr Asp Val Lys Gl - #u Leu Ile Pro Glu Phe     #      31650     - Phe Tyr Leu Pro Glu Phe Leu Val Asn Arg Gl - #u Gly Phe Asp Phe Gly     #  31805     - Val Arg Gln Asn Gly Glu Arg Val Asn His Va - #l Asn Leu Pro Pro Trp     #               32003190 - #                3195     - Ala Arg Asn Asp Pro Arg Leu Phe Ile Leu Il - #e His Arg Gln Ala Leu     #              32150     - Glu Ser Asp Tyr Val Ser Gln Asn Ile Cys Gl - #n Trp Ile Asp Leu Val     #          32305     - Phe Gly Tyr Lys Gln Lys Gly Lys Ala Ser Va - #l Gln Ala Ile Asn Val     #      32450     - Phe His Pro Ala Thr Tyr Phe Gly Met Asp Va - #l Ser Ala Val Glu Asp     #  32605     - Pro Val Gln Arg Arg Ala Leu Glu Thr Met Il - #e Lys Thr Tyr Gly Gln     #               32803270 - #                3275     - Thr Pro Arg Gln Leu Phe His Met Ala His Va - #l Ser Arg Pro Gly Ala     #              32950     - Lys Leu Asn Ile Glu Gly Glu Leu Pro Ala Al - #a Val Gly Leu Leu Val     #          33105     - Gln Phe Ala Phe Arg Glu Thr Arg Glu Gln Va - #l Lys Glu Ile Thr Tyr     #      33250     - Pro Ser Pro Leu Ser Trp Ile Lys Gly Leu Ly - #s Trp Gly Glu Tyr Val     #  33405     - Gly Ser Pro Ser Ala Pro Val Pro Val Val Cy - #s Phe Ser Gln Pro His     #               33603350 - #                3355     - Gly Glu Arg Phe Gly Ser Leu Gln Ala Leu Pr - #o Thr Arg Ala Ile Cys     #              33750     - Gly Leu Ser Arg Asn Phe Cys Leu Val Met Th - #r Tyr Ser Lys Glu Gln     #          33905     - Gly Val Arg Ser Met Asn Ser Thr Asp Ile Gl - #n Trp Ser Ala Ile Leu     #      34050     - Ser Trp Gly Tyr Ala Asp Asn Ile Leu Arg Le - #u Lys Ser Lys Gln Ser     #  34205     - Glu Pro Pro Val Asn Phe Ile Gln Ser Ser Gl - #n Gln Tyr Gln Val Thr     #               34403430 - #                3435     - Ser Cys Ala Trp Val Pro Asp Ser Cys Gln Le - #u Phe Thr Gly Ser Lys     #              34550     - Cys Gly Val Ile Thr Ala Tyr Thr Asn Arg Ph - #e Thr Ser Ser Thr Pro     #          34705     - Ser Glu Ile Glu Met Glu Thr Gln Ile His Le - #u Tyr Gly His Thr Glu     #      34850     - Glu Ile Thr Ser Leu Phe Val Cys Lys Pro Ty - #r Ser Ile Leu Ile Ser     #  35005     - Val Ser Arg Asp Gly Thr Cys Ile Ile Trp As - #p Leu Asn Arg Leu Cys     #               35203510 - #                3515     - Tyr Val Gln Ser Leu Ala Gly His Lys Ser Pr - #o Val Thr Ala Val Ser     #              35350     - Ala Ser Glu Thr Ser Gly Asp Ile Ala Thr Va - #l Cys Asp Ser Ala Gly     #          35505     - Gly Gly Ser Asp Leu Arg Leu Trp Thr Val As - #n Gly Asp Leu Val Gly     #      35650     - His Val His Cys Arg Glu Ile Ile Cys Ser Va - #l Ala Phe Ser Asn Gln     #  35805     - Pro Glu Gly Val Ser Ile Asn Val Ile Ala Gl - #y Gly Leu Glu Asn Gly     #               36003590 - #                3595     - Ile Val Arg Leu Trp Ser Thr Trp Asp Leu Ly - #s Pro Val Arg Glu Ile     #              36150     - Thr Phe Pro Lys Ser Asn Lys Pro Ile Ile Se - #r Leu Thr Phe Ser Cys     #          36305     - Asp Gly His His Leu Tyr Thr Ala Asn Ser As - #p Gly Thr Val Ile Ala     #      36450     - Trp Cys Arg Lys Asp Gln His Arg Leu Lys Gl - #n Pro Met Phe Tyr Ser     #  36605     - Phe Leu Ser Ser Tyr Ala Ala Gly     3665                3670     - (2) INFORMATION FOR SEQ ID NO:13:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 21 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:     #21                AGGC C     - (2) INFORMATION FOR SEQ ID NO:14:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 21 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:     #21                ACTA C     - (2) INFORMATION FOR SEQ ID NO:15:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 21 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:     #21                CAGA C     - (2) INFORMATION FOR SEQ ID NO:16:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 20 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:     # 20               TCTC     - (2) INFORMATION FOR SEQ ID NO:17:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 20 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:     # 20               AAAC     - (2) INFORMATION FOR SEQ ID NO:18:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 20 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:     # 20               GACT     - (2) INFORMATION FOR SEQ ID NO:19:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 20 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:     # 20               AAGT     - (2) INFORMATION FOR SEQ ID NO:20:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 20 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:     # 20               GTAG     - (2) INFORMATION FOR SEQ ID NO:21:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 20 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:     # 20               ATCC     - (2) INFORMATION FOR SEQ ID NO:22:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 20 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:     # 20               CATC     - (2) INFORMATION FOR SEQ ID NO:23:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 21 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:     #21                AATA G     - (2) INFORMATION FOR SEQ ID NO:24:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 21 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:     #21                TTAT G     - (2) INFORMATION FOR SEQ ID NO:25:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 20 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:     # 20               AGAC     - (2) INFORMATION FOR SEQ ID NO:26:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 20 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:     # 20               CAGA     - (2) INFORMATION FOR SEQ ID NO:27:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 22 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:     #                 22ATG CT     - (2) INFORMATION FOR SEQ ID NO:28:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 21 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:     #21                AGAT C     - (2) INFORMATION FOR SEQ ID NO:29:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 18 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:     #  18              GC     - (2) INFORMATION FOR SEQ ID NO:30:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 25 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:     #               25 GGGT GGTTA     - (2) INFORMATION FOR SEQ ID NO:31:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 21 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:     #21                AACT C     - (2) INFORMATION FOR SEQ ID NO:32:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 21 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:     #21                AGAA C     __________________________________________________________________________ 

What is claimed is:
 1. An isolated nucleic acid molecule comprising:(a) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO:2; (b) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO:10; or (c) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO:12.
 2. The isolated nucleic acid molecule of claim 1 wherein the nucleic acid molecule is a cDNA molecule.
 3. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises a nucleotide sequence which encodes the polypeptide shown in SEQ ID NO:2.
 4. The isolated nucleic acid molecule of claim 3 wherein the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:
 1. 5. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises a nucleotide sequence which encodes the polypeptide shown in SEQ ID NO:10.
 6. The isolated nucleic acid molecule of claim 5 wherein the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:9.
 7. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises a nucleotide sequence which encodes the polypeptide shown in SEQ ID NO:12.
 8. The isolated nucleic acid molecule of claim 7 wherein the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:11.
 9. A nucleotide vector comprising the nucleic acid molecule of claim
 1. 10. An expression vector comprising the nucleic acid molecule of claim 1 in operative association with a nucleotide regulatory sequence that controls expression of the nucleic acid in a host cell.
 11. An isolated host cell genetically engineered to contain the nucleic acid molecule of claim
 1. 12. An isolated host cell genetically engineered to contain the nucleic acid molecule of claim 1 in operative association with a regulatory sequence that controls expression of the nucleic acid in the host cell.
 13. An isolated nucleic acid molecule which comprises an allelic variant of a second nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO-9 or SEQ ID NO:11, wherein the nucleic acid molecule hybridizes under highly stringent conditions to the complement of the second nucleic acid molecule, said highly stringent conditions comprising hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% SDS, 1 mM EDTA at 65° C. and washing in 0.1×SSC/0.1% SDS at 68 ° C., and wherein the nucleic acid molecule is expressed at the same chromosomal locus as that expressing a nucleic acid molecule comprising SEQ ID NO: 1, SEQ ID NO:9 or SEQ ID NO-11.
 14. A nucleotide vector comprising the nucleic acid molecule of claim
 13. 15. An expression vector comprising the nucleic acid molecule of claim 5 in operative association with a nucleotide regulatory sequence that controls expression of the nucleic acid in a host cell.
 16. An isolated host cell genetically engineered to contain the nucleic acid molecule of claim
 13. 17. An isolated host cell genetically engineered to contain the nucleic acid molecule of claim 13 in operative association with a regulatory sequence that controls expression of the nucleic acid in the host cell.
 18. The isolated nucleic acid molecule of claim 13, wherein the nucleic acid molecule comprises an allelic variant of a second nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:
 1. 19. The isolated nucleic acid molecule of claim 13, wherein the nucleic acid molecule comprises an allelic variant of a second nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:9.
 20. The isolated nucleic acid molecule of claim 13, wherein the nucleic acid molecule comprises an allelic variant of a second nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO.
 11. 